Pth prodrugs

ABSTRACT

The present invention relates to PTH prodrugs, pharmaceutical compositions comprising such PTH prodrugs and their uses.

The present invention relates to PTH prodrugs, pharmaceuticalcompositions comprising such PTH prodrugs and their uses.

Hypoparathyroidism is a rare endocrine disease with low serum calciumand inappropriately low (insufficient) circulating parathyroid hormonelevels, most often in adults secondary to thyroid surgery. Standardtreatment includes activated vitamin D analogues and calciumsupplementation, which increases calcium and phosphorus absorption andserum levels at the expense of abnormally increased urinary calciumexcretion. Hypoparathyroidism is the only major endocrine conditiontoday, where the hormonal insufficiency in general is not treated bysubstitution of the missing hormone (PTH).

The prevalence of hypoparathyroidism has recently been systematicallystudied in Denmark, where a total of more than 2000 patients wereidentified giving a prevalence of ˜24/100 000 inhabitants, among whomonly a minority (2/100 000) had hypoparathyroidism due to non-surgicalcauses. These estimates are in agreement with recent data from the USA,showing a prevalence of the same magnitude for patients with chronichypoparathyroidism.

Endogenous PTH is synthesized and secreted by the parathyroid glands andis the principal endocrine hormone regulating systemic calcium andphosphorus homeostasis. Physiological actions of PTH include releasingcalcium and phosphorus from bone, retaining calcium but not phosphorusin the kidney by increasing renal tubular reabsorption of calcium butdecreasing renal tubular reabsorption of phosphate, and stimulating therenal production of active vitamin D (1,25(OH)₂vitamin D3) which in turnenhances intestinal calcium and phosphorus absorption. Without the renalactions of PTH to conserve calcium and excrete phosphorus, conventionaltherapy with vitamin D analogs and calcium supplementation may lead torenal insufficiency or failure due to progressive nephrocalcinosis, aswell as ectopic calcifications (in the basal ganglia, the lens of theeye, and the vascular system) due to a chronically increased calcium xphosphorus product, which precipitates out as calcium phosphate crystalswhen this product is maintained elevated for long periods.

Recently Natpara®, PTH(1-84) was approved by the FDA for the treatmentof hypoparathyroidism. Historically Forteo® PTH(1-34) has also been usedas once, twice or thrice daily injections for hypoparathyroidism,despite not being approved for this indication.

When PTH is delivered intermittently, such as by current daily ormultiple daily injections of PTH(1-84) or PTH(1-34) it acts on bone asan anabolic agent by preferentially activating osteoblasts overosteoclasts. This anabolic effect of intermittent PTH exposure contrastswith the net bone catabolism that can occur with continuous exposure toPTH. The anabolic potential of intermittent administration of PTHagonists has successfully been utilized for the treatment ofosteoporosis, where bone turnover is usually high and bone mineraldensity (BMD) is low, whereas the converse is the case forhypoparathyroidism.

A major complication of hypoparathyroidism is hypercalciuria, due to thelack of PTH dependent calcium reabsorption in the distal renal tubules.Hypercalciuria is associated with an increased risk of nephrocalcinosis,nephrolithiasis and kidney failure. According to the FDAs review ofNatpara, daily injections of PTH failed to provide adequate control ofurinary calcium excretion, due to the short half-life of this PTHagonist in the body.

Furthermore, unphysiological levels of PTH may be associated withhypercalcemia and hypocalcemia. Treatment with Natpara did not improvethe incidence of these complications compared to placebo. This can inpart be explained by the unfavorable PK of Natpara. For example,administration of the currently approved doses of Natpara results ingreatly supraphysiological levels of PTH with a C_(max) of 300 pg/ml,which returns to baseline at 12 hours. As a result patients are overtreated in the initial phase following administration and under treatedin the phase leading up to subsequent dosing.

Hypocalcemia is associated with numerous symptoms, some of which can belife threatening, including: tetany; paresthesias; impaired cognition;loss of consciousness with convulsions (grand mal seizures); impairedkidney function; heart arrhythmias and fainting, and even heart failure.

As such, there is a high unmet need for a more physiological PTHtherapy, providing a sustained exposure to PTH that enables alleviationof symptoms relating to hypocalcemia, hypercalciuria, andhyperphosphatemia, without causing hypercalcemia. PTH replacementtherapy would be more physiologic if delivered by continuous infusion,such as using an insulin pump. This has for example been demonstrated byWiner et al. (J Pediatr, 2014, 165(3), 556-563), where pump deliverysimultaneously normalized bone turnover markers and urine and serummineral levels, whereas intermittent injection delivery did not.

The normal PTH range is 15-50 pg/ml, and it is important to appreciatethat intermittent PTH agonist administration does not constitutephysiological replacement therapy. PTH polypeptides have inherentlyshort circulating half-lives because of rapid hepatic metabolism. Therapid clearance of the drug from the body prevents sufficient drugcoverage throughout the dosing interval, despite initialsupraphysiological drug levels.

Several approaches have been applied to create longer acting version ofPTH, including encapsulation of PTH in PLGA microparticles and permanentconjugation of the PTH molecule to either synthetic or peptidicpolymers. Kostenuik et al. (J Bone Miner Res, 2007, 22(10), 1534-1547),described a PTH-Fc fusion protein with a longer half-life than PTH(1-34)and studies were conducted in osteopenic ovariectomized rats and mice todetermine whether intermittent (one to two per week) injections ofPTH-Fc would increase bone mass, density, and strength despite theprolonged duration of exposure to PTH. It was demonstrated that aPTH-derived molecule with a sustained circulating half-life providedcomparable anabolic effects on cortical and cancellous bone to dailyPTH, but with a reduced dosing frequency. Another approach has beensuggested by Ponnapakkam et al. (Drug Discov Today, 2014, 19(3),204.208), in which a hybrid polypeptide of PTH and a collagen bindingdomain caused long-term (up to 12 months) increases in bone mineraldensity in normal female mice after a single dose.

Patients with hypoparathyroidism typically demonstrate an abnormally lowrate of bone turnover resulting in increased bone mineral density, andas such the anabolic effects of PTH should be avoided when treatingHypoparathyroidism, and optimally treatment should normalize their rateof bone turnover, but not increase it to above the normal range, as hasbeen demonstrated with daily treatment with PTH(1-34) and PTH(1-84).

In summary, there is a need for a more efficacious PTH treatment.

It is therefore an object of the present invention to at least partiallyovercome the shortcomings described above.

This object is achieved with a PTH prodrug or a pharmaceuticallyacceptable salt thereof, wherein the prodrug is of formula (Ia) or (Ib)

Z-(-L²-L¹-D)_(x)  (Ia)

D-(-L¹-L²-Z)_(y)  (Ib),

-   -   wherein        -   D is a PTH moiety;        -   L¹- is a reversible prodrug linker moiety connected to the            PTH moiety -D through a functional group of PTH;        -   L²- is a single chemical bond or a spacer moiety;        -   Z is a water-soluble carrier moiety;        -   x is an integer selected from the group consisting of 1, 2,            3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; and        -   y is an integer selected from the group consisting of 1, 2,            3, 4 and 5.

It was surprisingly found that the PTH prodrugs of the present inventionexhibit a low residual activity of the prodrug and provide a sustainedrelease of PTH. As a result, administration of the PTH prodrugs of thepresent invention leads to a concurrent normalization of serum calciumand a reduction in serum phosphate and thus to an increased serumcalcium to serum phosphate ratio compared to treatment with PTH1-84, thecurrent standard of care. At the same time no adverse effects on boneresorption and formation markers and overall bone health in the relevantanimal model for the human condition were observed upon administrationof physiological doses.

It was also surprisingly found that such PTH prodrugs are capable ofachieving a stable plasma profile of PTH which ensures physiologicalserum and urinary calcium levels or even lower than normal urinarycalcium levels.

Within the present invention the terms are used having the meaning asfollows.

As used herein the term “PTH” refers to all PTH polypeptides, preferablyfrom mammalian species, more preferably from human and mammalianspecies, more preferably from human and murine species, as well as theirvariants, analogs, orthologs, homologs, and derivatives and fragmentsthereof, that are characterized by raising serum calcium and renalphosphorus excretion, and lowering serum phosphorus and renal calciumexcretion. The term “PTH” also refers to all PTHrP polypeptides, such asthe polypeptide of SEQ ID NO:121, that bind to and activate the commonPTH/PTHrP1 receptor. Preferably, the term “PTH” refers to the PTHpolypeptide of SEQ ID NO:51 as well as its variants, homologs andderivatives exhibiting essentially the same biological activity, i.e.raising serum calcium and renal phosphorus excretion, and lowering serumphosphorus and renal calcium excretion.

Preferably, the term “PTH” refers to the following polypeptidesequences:

(PTH 1-84) SEQ ID NO: 1SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (PTH 1-83) SEQ ID NO: 2SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKS (PTH 1-82) SEQ ID NO: 3SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAK (PTH 1-81) SEQ ID NO: 4SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKA (PTH 1-80) SEQ ID NO: 5SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTK (PTH 1-79) SEQ ID NO: 6SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLT (PTH 1-78) SEQ ID NO: 7SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVL (PTH 1-77) SEQ ID NO: 8SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNV (PTH 1-76) SEQ ID NO: 9SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVN (PTH 1-75) SEQ ID NO: 10SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADV (PTH 1-74) SEQ ID NO: 11SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKAD (PTH 1-73) SEQ ID NO: 12SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKA (PTH 1-72) SEQ ID NO: 13SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADK (PTH 1-71) SEQ ID NO: 14SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKSLGEAD(PTH 1-70) SEQ ID NO: 15SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKSLGEA(PTH 1-69) SEQ ID NO: 16SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKSLGE(PTH 1-68) SEQ ID NO: 17SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKSLG(PTH 1-67) SEQ ID NO: 18SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKSL(PTH 1-66) SEQ ID NO: 19SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEKS(PTH 1-65) SEQ ID NO: 20SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHEK(PTH 1-64) SEQ ID NO: 21SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESHE(PTH 1-63) SEQ ID NO: 22SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVESH(PTH 1-62) SEQ ID NO: 23SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVES(PTH 1-61) SEQ ID NO: 24SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLVE(PTH 1-60) SEQ ID NO: 25SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNVLV (PTH 1-59)SEQ ID NO: 26 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVL (PTH 1-58) SEQ ID NO: 27SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDNV (PTH 1-57)SEQ ID NO: 28 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKEDN(PTH 1-56) SEQ ID NO: 29SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKED (PTH 1-55)SEQ ID NO: 30 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKKE(PTH 1-54) SEQ ID NO: 31SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRKK (PTH 1-53)SEQ ID NO: 32 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PRK(PTH 1-52) SEQ ID NO: 33SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR PR (PTH 1-51)SEQ ID NO: 34 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR P(PTH 1-50) SEQ ID NO: 35SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR (PTH 1-49)SEQ ID NO: 36 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ(PTH 1-48) SEQ ID NO: 37SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS (PTH 1-47)SEQ ID NO: 38 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG (PTH 1-46)SEQ ID NO: 39 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDA (PTH 1-45)SEQ ID NO: 40 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRD (PTH 1-44)SEQ ID NO: 41 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR (PTH 1-43)SEQ ID NO: 42 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAP (PTH 1-42)SEQ ID NO: 43 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA (PTH 1-41)SEQ ID NO: 44 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL (PTH 1-40)SEQ ID NO: 45 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP (PTH 1-39)SEQ ID NO: 46 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA (PTH 1-38)SEQ ID NO: 47 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG (PTH 1-37)SEQ ID NO: 48 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL (PTH 1-36)SEQ ID NO: 49 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA (PTH 1-35)SEQ ID NO: 50 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV (PTH 1-34)SEQ ID NO: 51 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (PTH 1-33)SEQ ID NO: 52 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN (PTH 1-32) SEQ ID NO: 53SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH (PTH 1-31) SEQ ID NO: 54SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV (PTH 1-30) SEQ ID NO: 55SVSEIQLMHNLGKHLNSMERVEWLRKKLQD (PTH 1-29) SEQ ID NO: 56SVSEIQLMHNLGKHLNSMERVEWLRKKLQ (PTH 1-28) SEQ ID NO: 57SVSEIQLMHNLGKHLNSMERVEWLRKKL (PTH 1-27) SEQ ID NO: 58SVSEIQLMHNLGKHLNSMERVEWLRKK (PTH 1-26) SEQ ID NO: 59SVSEIQLMHNLGKHLNSMERVEWLRK (PTH 1-25) SEQ ID NO: 60SVSEIQLMHNLGKHLNSMERVEWLR (amidated PTH 1-84) SEQ ID NO: 61SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ; wherein the  C-terminus is amidated(amidated PTH 1-83) SEQ ID NO: 62SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKS; wherein the  C-terminus is amidated(amidated PTH 1-82) SEQ ID NO: 63SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAK; wherein the  C-terminus is amidated(amidated PTH 1-81) SEQ ID NO: 64SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKA; wherein the  C-terminus is amidated(amidated PTH 1-80) SEQ ID NO: 65SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTK; wherein the  C-terminus is amidated(amidated PTH 1-79) SEQ ID NO: 66SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLT; wherein the  C-terminus is amidated(amidated PTH 1-78) SEQ ID NO: 67SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVL; wherein the  C-terminus is amidated(amidated PTH 1-77) SEQ ID NO: 68SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNV; wherein the  C-terminus is amidated(amidated PTH 1-76) SEQ ID NO: 69SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVN; wherein the  C-terminus is amidated(amidated PTH 1-75) SEQ ID NO: 70SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADV; wherein the C-terminus is amidated(amidated PTH 1-74) SEQ ID NO: 71SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKAD; wherein the C-terminus is amidated(amidated PTH 1-73) SEQ ID NO: 72SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKA; wherein the C-terminus is amidated(amidated PTH 1-72) SEQ ID NO: 73SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADK; wherein the C-terminus is amidated(amidated PTH 1-71) SEQ ID NO: 74SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEAD; wherein the C-terminus is amidated(amidated PTH 1-70) SEQ ID NO: 75SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEA; wherein the C-terminus is amidated(amidated PTH 1-69) SEQ ID NO: 76SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGE; wherein the C-terminus is amidated(amidated PTH 1-68) SEQ ID NO: 77SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLG; wherein the C-terminus is amidated(amidated PTH 1-67) SEQ ID NO: 78SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSL; wherein the C-terminus is amidated(amidated PTH 1-66) SEQ ID NO: 79SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKS; wherein the C-terminus is amidated (amidated PTH 1-65)SEQ ID NO: 80 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEK; wherein the C-terminus is amidated (amidated PTH 1-64)SEQ ID NO: 81 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHE; wherein the C-terminus is amidated (amidated PTH 1-63)SEQ ID NO: 82 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESH; wherein the C-terminus is amidated (amidated PTH 1-62)SEQ ID NO: 83 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVES; wherein the C-terminus is amidated (amidated PTH 1-61)SEQ ID NO: 84 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVE; wherein the C-terminus is amidated (amidated PTH 1-60)SEQ ID NO: 85 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLV; wherein the C-terminus is amidated (amidated PTH 1-59)SEQ ID NO: 86 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVL; wherein the C-terminus is amidated (amidated PTH 1-58)SEQ ID NO: 87 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNV; wherein the C-terminus is amidated (amidated PTH 1-57)SEQ ID NO: 88 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDN; wherein the C-terminus is amidated (amidated PTH 1-56)SEQ ID NO: 89 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKED; wherein the C-terminus is amidated (amidated PTH 1-55)SEQ ID NO: 90 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKE; wherein the C-terminus is amidated amidated (PTH 1-54)SEQ ID NO: 91 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKK; wherein the C-terminus is amidated (amidated PTH 1-53)SEQ ID NO: 92 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRK; wherein the C-terminus is amidated (amidated PTH 1-52)SEQ ID NO: 93 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPR; wherein the C-terminus is amidated (amidated PTH 1-51) SEQ ID NO: 94SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRP; wherein the C-terminus is amidated (amidated PTH 1-50) SEQ ID NO: 95SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR; wherein the C-terminus is amidated (amidated PTH 1-49)SEQ ID NO: 96 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ;wherein the C-terminus is amidated (amidated PTH 1-48) SEQ ID NO: 97SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS;wherein the C-terminus is amidated (amidated PTH 1-47) SEQ ID NO: 98SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG;wherein the C-terminus is amidated (amidated PTH 1-46) SEQ ID NO: 99SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDA;wherein the C-terminus is amidated (amidated PTH 1-45) SEQ ID NO: 100SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRD;wherein the C-terminus is amidated (amidated PTH 1-44) SEQ ID NO: 101SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR;wherein the C-terminus is amidated (amidated PTH 1-43) SEQ ID NO: 102SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAP;wherein the C-terminus is amidated (amidated PTH 1-42) SEQ ID NO: 103SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA;wherein the C-terminus is amidated (amidated PTH 1-41) SEQ ID NO: 104SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL; whereinthe C-terminus is amidated (amidated PTH 1-40) SEQ ID NO: 105SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP; whereinthe C-terminus is amidated (amidated PTH 1-39) SEQ ID NO: 106SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA; whereinthe C-terminus is amidated (amidated PTH 1-38) SEQ ID NO: 107SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG; whereinthe C-terminus is amidated (amidated PTH 1-37) SEQ ID NO: 108SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL; wherein theC-terminus is amidated (amidated PTH 1-36) SEQ ID NO: 109SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA; wherein the C-terminus is amidated(amidated PTH 1-35) SEQ ID NO: 110SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV; wherein the C-terminus is amidated(amidated PTH 1-34) SEQ ID NO: 111SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF; wherein the C-terminus is amidated(amidated PTH 1-33) SEQ ID NO: 112SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN; wherein the C-terminus is amidated(amidated PTH 1-32) SEQ ID NO: 113SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH; wherein the C-terminus is amidated(amidated PTH 1-31) SEQ ID NO: 114SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV; wherein the C-terminus is amidated(amidated PTH 1-30) SEQ ID NO: 115SVSEIQLMHNLGKHLNSMERVEWLRKKLQD; wherein the C-terminus is amidated(amidated PTH 1-29) SEQ ID NO: 116SVSEIQLMHNLGKHLNSMERVEWLRKKLQ; wherein the C-terminus is amidated(amidated PTH 1-28) SEQ ID NO: 117SVSEIQLMHNLGKHLNSMERVEWLRKKL; wherein the C-terminus is amidated(amidated PTH 1-27) SEQ ID NO: 118SVSEIQLMHNLGKHLNSMERVEWLRKK; wherein the C-terminus is amidated(amidated PTH 1-26) SEQ ID NO: 119SVSEIQLMHNLGKHLNSMERVEWLRK; wherein the C-terminus is amidated(amidated PTH 1-25) SEQ ID NO: 120SVSEIQLMHNLGKHLNSMERVEWLR; wherein the C-terminus is amidated (PTH rP)SEQ ID NO: 121 AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRRH

More preferably, the term “PTH” refers to the sequence of SEQ ID:NOs 47,48, 49, 50, 51, 52, 53, 54, 55, 107, 108, 109, 110, 111, 112, 113, 114and 115. Even more preferably, the term “PTH” refers to the sequence ofSEQ ID:NOs 50, 51, 52, 110, 111 and 112. In a particularly preferredembodiment the term “PTH” refers to the sequence of SEQ ID NO:51.

As used herein, the term “PTH polypeptide variant” refers to apolypeptide from the same species that differs from a reference PTH orPTHrP polypeptide. Preferably, such reference is a PTH polypeptidesequence and has the sequence of SEQ ID NO:51. Generally, differencesare limited so that the amino acid sequence of the reference and thevariant are closely similar overall and, in many regions, identical.Preferably, PTH polypeptide variants are at least 70%, 80%, 90%, or 95%identical to a reference PTH or PTHrP polypeptide, preferably to the PTHpolypeptide of SEQ ID NO:51. By a polypeptide having an amino acidsequence at least, for example, 95% “identical” to a query amino acidsequence, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. These alterationsof the reference sequence may occur at the amino (N-terminal) or carboxyterminal (C-terminal) positions of the reference amino acid sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. The query sequence maybe an entire amino acid sequence of the reference sequence or anyfragment specified as described herein. Preferably, the query sequenceis the sequence of SEQ ID NO:51.

Such PTH polypeptide variants may be naturally occurring variants, suchas naturally occurring allelic variants encoded by one of severalalternate forms of a PTH or PTHrP occupying a given locus on achromosome or an organism, or isoforms encoded by naturally occurringsplice variants originating from a single primary transcript.Alternatively, a PTH polypeptide variant may be a variant that is notknown to occur naturally and that can be made by mutagenesis techniquesknown in the art.

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus of a bioactive polypeptide withoutsubstantial loss of biological function. Such N- and/or C-terminaldeletions are also encompassed by the term PTH polypeptide variant.

It is also recognized by one of ordinary skill in the art that someamino acid sequences of PTH or PTHrP polypeptides can be varied withoutsignificant effect of the structure or function of the polypeptide. Suchmutants include deletions, insertions, inversions, repeats, andsubstitutions selected according to general rules known in the art so asto have little effect on activity. For example, guidance concerning howto make phenotypically silent amino acid substitutions is provided inBowie et al. (1990), Science 247:1306-1310, which is hereby incorporatedby reference in its entirety, wherein the authors indicate that thereare two main approaches for studying the tolerance of the amino acidsequence to change.

The term PTH polypeptide also encompasses all PTH and PTHrP polypeptidesencoded by PTH and PTHrP analogs, orthologs, and/or species homologs. Itis also recognized by one of ordinary skill in the art that PTHrP andPTHrP analogs bind to activate the common PTH/PTHrP1 receptor, so theterm PTH polypeptide also encompasses all PTHrP analogs. As used herein,the term “PTH analog” refers to PTH and PTHrP of different and unrelatedorganisms which perform the same functions in each organism but whichdid not originate from an ancestral structure that the organisms'ancestors had in common. Instead, analogous PTH and PTHrP aroseseparately and then later evolved to perform the same or similarfunctions. In other words, analogous PTH and PTHrP polypeptides arepolypeptides with quite different amino acid sequences but that performthe same biological activity, namely raising serum calcium and renalphosphorus excretion, and lowering serum phosphorus and renal calciumexcretion.

As used herein the term “PTH ortholog” refers to PTH and PTHrP withintwo different species which sequences are related to each other via acommon homologous PTH or PTHrP in an ancestral species, but which haveevolved to become different from each other.

As used herein, the term “PTH homolog” refers to PTH and PTHrP ofdifferent organisms which perform the same functions in each organismand which originate from an ancestral structure that the organisms'ancestors had in common. In other words, homologous PTH polypeptides arepolypeptides with quite similar amino acid sequences that perform thesame biological activity, namely raising serum calcium and renalphosphorus excretion, and lowering serum phosphorus and renal calciumexcretion. Preferably, PTH polypeptide homologs may be defined aspolypeptides exhibiting at least 40%, 50%, 60%, 70%, 80%, 90% or 95%identity to a reference PTH or PTHrP polypeptide, preferably the PTHpolypeptide of SEQ ID NO:51.

Thus, a PTH polypeptide according to the invention may be, for example:(i) one in which at least one of the amino acids residues is substitutedwith a conserved or non-conserved amino acid residue, preferably aconserved amino acid residue, and such substituted amino acid residuemay or may not be one encoded by the genetic code; and/or (ii) one inwhich at least one of the amino acid residues includes a substituentgroup; and/or (iii) one in which the PTH polypeptide is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol); and/or (iv) one in whichadditional amino acids are fused to the PTH polypeptide, such as an IgGFc fusion region polypeptide or leader or secretory sequence or asequence which is employed for purification of the above form of thepolypeptide or a pre-protein sequence.

As used herein, the term “PTH polypeptide fragment” refers to anypolypeptide comprising a contiguous span of a part of the amino acidsequence of a PTH or PTHrP polypeptide, preferably the polypeptide ofSEQ ID NO:51.

More specifically, a PTH polypeptide fragment comprises at least 6, suchas at least 8, at least 10 or at least 17 consecutive amino acids of aPTH or PTHrP polypeptide, more preferably of the polypeptide of SEQ IDNO:51. A PTH polypeptide fragment may additionally be described assub-genuses of PTH or PTHrP polypeptides comprising at least 6 aminoacids, wherein “at least 6” is defined as any integer between 6 and theinteger representing the C-terminal amino acid of a PTH or PTHrPpolypeptide, preferably of the polypeptide of SEQ ID No:51. Furtherincluded are species of PTH or PTHrP polypeptide fragments at least 6amino acids in length, as described above, that are further specified interms of their N-terminal and C-terminal positions. Also encompassed bythe term “PTH polypeptide fragment” as individual species are all PTH orPTHrP polypeptide fragments, at least 6 amino acids in length, asdescribed above, that may be particularly specified by a N-terminal andC-terminal position. That is, every combination of a N-terminal andC-terminal position that a fragment at least 6 contiguous amino acidresidues in length could occupy, on any given amino acid sequence of aPTH or PTHrP polypeptide, preferably the PTH polypeptide of SEQ ID:NO51,is included in the present invention.

The term “PTH” also includes poly(amino acid) conjugates which have asequence as described above, but having a backbone that comprises bothamide and non-amide linkages, such as ester linkages, like for exampledepsipeptides. Depsipeptides are chains of amino acid residues in whichthe backbone comprises both amide (peptide) and ester bonds.Accordingly, the term “side chain” as used herein refers either to themoiety attached to the alpha-carbon of an amino acid moiety, if theamino acid moiety is connected through amine bonds such as inpolypeptides, or to any carbon atom-comprising moiety attached to thebackbone of a poly(amino acid) conjugate, such as for example in thecase of depsipeptides. Preferably, the term “PTH” refers to polypeptideshaving a backbone formed through amide (peptide) bonds.

As the term PTH includes the above-described variants, analogs,orthologs, homologs, derivatives and fragments of PTH and PTHrP, allreferences to specific positions within a reference sequence alsoinclude the equivalent positions in variants, analogs, orthologs,homologs, derivatives and fragments of a PTH or PTHrP moiety, even ifnot specifically mentioned.

As used herein, the term “random coil” refers to a peptide or proteinadopting/having/forming, preferably having, a conformation whichsubstantially lacks a defined secondary and tertiary structure asdetermined by circular dichroism spectroscopy performed in aqueousbuffer at ambient temperature, and pH 7.4. Preferably, ambienttemperature is about 20° C., i.e. between 18° C. and 22° C., mostpreferably ambient temperature is 20° C.

As used herein the term “pharmaceutical composition” refers to acomposition containing one or more active ingredients, for example adrug or a prodrug, here specifically the PTH prodrugs of the presentinvention, and optionally one or more excipients, as well as any productwhich results, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients of the composition, orfrom dissociation of one or more of the ingredients, or from other typesof reactions or interactions of one or more of the ingredients.Accordingly, the pharmaceutical compositions of the present inventionencompass any composition made by admixing one or more PTH prodrugs ofthe present invention and optionally a pharmaceutically acceptableexcipient.

As used herein the term “liquid composition” refers to a mixturecomprising water-soluble PTH prodrug and one or more solvents, such aswater.

The term “suspension composition” relates to a mixture comprisingwater-insoluble PTH prodrug, where for example the carrier Z′ is ahydrogel, and one or more solvents, such as water. Due to thewater-insoluble polymer, the polymeric prodrug cannot dissolve andrenders the prodrug in a particulate state.

As used herein, the term “dry composition” means that a pharmaceuticalcomposition is provided in a dry form. Suitable methods for drying arespray-drying and lyophilization, i.e. freeze-drying. Such drycomposition of prodrug has a residual water content of a maximum of 10%,preferably less than 5% and more preferably less than 2%, determinedaccording to Karl Fischer. Preferably, the pharmaceutical composition ofthe present invention is dried by lyophilization.

The term “drug” as used herein refers to a substance used in thetreatment, cure, prevention, or diagnosis of a disease or used tootherwise enhance physical or mental well-being. If a drug is conjugatedto another moiety, the moiety of the resulting product that originatedfrom the drug is referred to as “biologically active moiety”. The PTHprodrug of the present invention comprise a PTH moiety which is releasedfrom the PTH prodrug in the form of the drug PTH.

As used herein the term “prodrug” refers to a conjugate comprising abiologically active moiety reversibly and covalently connected to aspecialized protective group through a reversible prodrug linker moietywhich is a linker moiety comprising a reversible linkage with thebiologically active moiety and wherein the specialized protective groupalters or eliminates undesirable properties in the parent molecule. Thisalso includes the enhancement of desirable properties in the drug andthe suppression of undesirable properties. The specialized non-toxicprotective group is referred to as “carrier”. A prodrug releases thereversibly and covalently bound biologically active moiety in the formof its corresponding drug. In other words, a prodrug is a conjugatecomprising a biologically active moiety which is covalently andreversibly conjugated to a carrier moiety via a reversible prodruglinker moiety, which covalent and reversible conjugation of the carrierto the reversible prodrug linker moiety is either directly or through aspacer. Such conjugate releases the formerly conjugated biologicallyactive moiety in the form of a free drug.

A “biodegradable linkage” or a “reversible linkage” is a linkage that ishydrolytically degradable, i.e. cleavable, in the absence of enzymesunder physiological conditions (aqueous buffer at pH 7.4, 37° C.) with ahalf-life ranging from one hour to two months, preferably from one hourto one month. Accordingly, a stable linkage is a linkage having ahalf-life under physiological conditions (aqueous buffer at pH 7.4, 37°C.) of more than two months.

Accordingly, a “reversible prodrug linker moiety” is a moiety which iscovalently conjugated to a biologically active moiety, such as PTH,through a reversible linkage and is also covalently conjugated to acarrier moiety, such as —Z or —Z′, wherein the covalent conjugation tosaid carrier moiety is either directly or through a spacer moiety, suchas -L²-. Preferably the linkage between —Z or —Z′ and -L²- is a stablelinkage.

As used herein, the term “traceless prodrug linker” means a reversibleprodrug linker which upon cleavage releases the drug in its free form.As used herein, the term “free form” of a drug means the drug in itsunmodified, pharmacologically active form.

As used herein, the term “excipient” refers to a diluent, adjuvant, orvehicle with which the therapeutic, such as a drug or prodrug, isadministered. Such pharmaceutical excipient can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, including but not limited to peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred excipientwhen the pharmaceutical composition is administered orally. Saline andaqueous dextrose are preferred excipients when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions are preferably employed as liquidexcipients for injectable solutions. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The pharmaceuticalcomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, pH buffering agents, like, for example, acetate,succinate, tris, carbonate, phosphate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid), or can contain detergents, likeTween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids like, forexample, glycine, lysine, or histidine. These pharmaceuticalcompositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, sustained-release formulations andthe like. The pharmaceutical composition can be formulated as asuppository, with traditional binders and excipients such astriglycerides. Oral formulation can include standard excipients such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Suchcompositions will contain a therapeutically effective amount of the drugor biologically active moiety, together with a suitable amount ofexcipient so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

As used herein, the term “reagent” means a chemical compound whichcomprises at least one functional group for reaction with the functionalgroup of another chemical compound or drug. It is understood that a drugcomprising a functional group (such as a primary or secondary amine orhydroxyl functional group) is also a reagent.

As used herein, the term “moiety” means a part of a molecule, whichlacks one or more atom(s) compared to the corresponding reagent. If, forexample, a reagent of the formula “H—X—H” reacts with another reagentand becomes part of the reaction product, the corresponding moiety ofthe reaction product has the structure “H—X—” or “—X—”, whereas each “-”indicates attachment to another moiety. Accordingly, a biologicallyactive moiety is released from a prodrug as a drug.

It is understood that if the sequence or chemical structure of a groupof atoms is provided which group of atoms is attached to two moieties oris interrupting a moiety, said sequence or chemical structure can beattached to the two moieties in either orientation, unless explicitlystated otherwise. For example, a moiety “—C(O)N(R)—” can be attached totwo moieties or interrupting a moiety either as “—C(O)N(R)—” or as“—N(R)C(O)—”. Similarly, a moiety

can be attached to two moieties or can interrupt a moiety either as

As used herein, the term “functional group” means a group of atoms whichcan react with other groups of atoms. Functional groups include but arenot limited to the following groups: carboxylic acid (—(C═O)OH), primaryor secondary amine (—NH₂, —NH—), maleimide, thiol (—SH), sulfonic acid(—(O═S=O)OH), carbonate, carbamate (—O(C═O)N<), hydroxyl (—OH), aldehyde(—(C═O)H), ketone (—(C═O)—), hydrazine (>N—N<), isocyanate,isothiocyanate, phosphoric acid (—O(P═O)OHOH), phosphonic acid(—O(P═O)OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride,hydroxylamine, disulfide, sulfonamides, sulfuric acid, vinyl sulfone,vinyl ketone, diazoalkane, oxirane, and aziridine.

In case the prodrugs of the present invention comprise one or moreacidic or basic groups, the invention also comprises their correspondingpharmaceutically or toxicologically acceptable salts, in particulartheir pharmaceutically utilizable salts. Thus, the prodrugs of thepresent invention comprising acidic groups can be used according to theinvention, for example, as alkali metal salts, alkaline earth metalsalts or as ammonium salts. More precise examples of such salts includesodium salts, potassium salts, calcium salts, magnesium salts or saltswith ammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Prodrugs of the presentinvention comprising one or more basic groups, i.e. groups which can beprotonated, can be present and can be used according to the invention inthe form of their addition salts with inorganic or organic acids.Examples for suitable acids include hydrogen chloride, hydrogen bromide,phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, aceticacid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formicacid, propionic acid, pivalic acid, diethylacetic acid, malonic acid,succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid,isonicotinic acid, citric acid, adipic acid, and other acids known tothe person skilled in the art. For the person skilled in the art furthermethods are known for converting the basic group into a cation like thealkylation of an amine group resulting in a positively-charge ammoniumgroup and an appropriate counterion of the salt. If the prodrugs of thepresent invention simultaneously comprise acidic and basic groups, theinvention also includes, in addition to the salt forms mentioned, innersalts or betaines (zwitterions). The respective salts can be obtained bycustomary methods which are known to the person skilled in the art like,for example by contacting these prodrugs with an organic or inorganicacid or base in a solvent or dispersant, or by anion exchange or cationexchange with other salts. The present invention also includes all saltsof the prodrugs of the present invention which, owing to lowphysiological compatibility, are not directly suitable for use inpharmaceuticals but which can be used, for example, as intermediates forchemical reactions or for the preparation of pharmaceutically acceptablesalts.

The term “pharmaceutically acceptable” means a substance that does causeharm when administered to a patient and preferably means approved by aregulatory agency, such as the EMA (Europe) and/or the FDA (US) and/orany other national regulatory agency for use in animals, preferably foruse in humans.

As used herein the term “about” in combination with a numerical value isused to indicate a range ranging from and including the numerical valueplus and minus no more than 10% of said numerical value, more preferablyno more than 8% of said numerical value, even more preferably no morethan 5% of said numerical value and most preferably no more than 2% ofsaid numerical value. For example, the phrase “about 200” is used tomean a range ranging from and including 200+/−10%, i.e. ranging from andincluding 180 to 220; preferably 200+/−8%, i.e. ranging from andincluding 184 to 216; even more preferably ranging from and including200+/−5%, i.e. ranging from and including 190 to 210; and mostpreferably 200+/−2%, i.e. ranging from and including 196 to 204. It isunderstood that a percentage given as “about 20%” does not mean“20%+/−10%”, i.e. ranging from and including 10 to 30%, but “about 20%”means ranging from and including 18 to 22%, i.e. plus and minus 10% ofthe numerical value which is 20.

As used herein, the term “polymer” means a molecule comprising repeatingstructural units, i.e. the monomers, connected by chemical bonds in alinear, circular, branched, crosslinked or dendrimeric way or acombination thereof, which may be of synthetic or biological origin or acombination of both. It is understood that a polymer may also compriseone or more other chemical group(s) and/or moiety/moieties, such as, forexample, one or more functional group(s). Preferably, a soluble polymerhas a molecular weight of at least 0.5 kDa, e.g. a molecular weight ofat least 1 kDa, a molecular weight of at least 2 kDa, a molecular weightof at least 3 kDa or a molecular weight of at least 5 kDa. If thepolymer is soluble, it preferable has a molecular weight of at most 1000kDa, such as at most 750 kDa, such as at most 500 kDa, such as at most300 kDa, such as at most 200 kDa, such as at most 100 kDa. It isunderstood that for insoluble polymers, such as hydrogels, no meaningfulmolecular weight ranges can be provided. It is understood that also aprotein is a polymer in which the amino acids are the repeatingstructural units, even though the side chains of each amino acid may bedifferent.

As used herein, the term “polymeric” means a reagent or a moietycomprising one or more polymer(s) or polymer moiety/moieties. Apolymeric reagent or moiety may optionally also comprise one or moreother moiety/moieties, which are preferably selected from the groupconsisting of:

-   -   C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-        to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,        phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and    -   linkages selected from the group comprising

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent, and —R and —R^(a) are independently of each other        selected from the group consisting of —H, methyl, ethyl, propyl,        butyl, pentyl and hexyl.

The person skilled in the art understands that the polymerizationproducts obtained from a polymerization reaction do not all have thesame molecular weight, but rather exhibit a molecular weightdistribution. Consequently, the molecular weight ranges, molecularweights, ranges of numbers of monomers in a polymer and numbers ofmonomers in a polymer as used herein, refer to the number averagemolecular weight and number average of monomers, i.e. to the arithmeticmean of the molecular weight of the polymer or polymeric moiety and thearithmetic mean of the number of monomers of the polymer or polymericmoiety.

Accordingly, in a polymeric moiety comprising “x” monomer units anyinteger given for “x” therefore corresponds to the arithmetic meannumber of monomers. Any range of integers given for “x” provides therange of integers in which the arithmetic mean numbers of monomers lies.An integer for “x” given as “about x” means that the arithmetic meannumbers of monomers lies in a range of integers of x+/−10%, preferablyx+/−8%, more preferably x+/−5% and most preferably x+/−2%.

As used herein, the term “number average molecular weight” means theordinary arithmetic mean of the molecular weights of the individualpolymers.

As used herein the term “water-soluble” with reference to a carriermeans that when such carrier is part of the PTH prodrug of the presentinvention at least 1 g of the PTH prodrug comprising such water-solublecarrier can be dissolved in one liter of water at 20° C. to form ahomogeneous solution. Accordingly, the term “water-insoluble” withreference to a carrier means that when such carrier is part of the PTHprodrug of the present invention less than 1 g of the PTH prodrugcomprising such water-insoluble carrier can be dissolved in one liter ofwater at 20° C. to form a homogeneous solution.

As used herein, the term “hydrogel” means a hydrophilic or amphiphilicpolymeric network composed of homopolymers or copolymers, which isinsoluble due to the presence of hydrogen bonds, ionic interactionsand/or covalent chemical crosslinks. The crosslinks provide the networkstructure and physical integrity.

As used herein the term “thermogelling” means a compound that is aliquid or a low viscosity solution having a viscosity of less than 500cps at 25° C. at a shear rate of about 0.1/second at a low temperature,which low temperature ranges between about 0° C. to about 10° C., butwhich is a higher viscosity compound of less than 10000 cps at 25° C. ata shear rate of about 0.1/second at a higher temperature, which highertemperature ranges between about 30° C. to about 40° C., such as atabout 37° C.

As used herein, the term “PEG-based” in relation to a moiety or reagentmeans that said moiety or reagent comprises PEG. Preferably, a PEG-basedmoiety or reagent comprises at least 10% (w/w) PEG, such as at least 20%(w/w) PEG, such as at least 30% (w/w) PEG, such as at least 40% (w/w)PEG, such as at least 50% (w/w), such as at least 60 (w/w) PEG, such asat least 70% (w/w) PEG, such as at least 80% (w/w) PEG, such as at least90% (w/w) PEG, such as at least 95%. The remaining weight percentage ofthe PEG-based moiety or reagent are other moieties preferably selectedfrom the following moieties and linkages:

-   -   C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-        to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,        phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and    -   linkages selected from the group comprising

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent, and —R and —R^(a) are independently of each other        selected from the group consisting of —H, methyl, ethyl, propyl,        butyl, pentyl and hexyl.

As used herein, the term “PEG-based comprising at least X % PEG” inrelation to a moiety or reagent means that said moiety or reagentcomprises at least X % (w/w) ethylene glycol units (—CH₂CH₂O—), whereinthe ethylene glycol units may be arranged blockwise, alternating or maybe randomly distributed within the moiety or reagent and preferably allethylene glycol units of said moiety or reagent are present in oneblock; the remaining weight percentage of the PEG-based moiety orreagent are other moieties preferably selected from the followingmoieties and linkages:

-   -   C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-        to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,        phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and    -   linkages selected from the group comprising

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent, and —R and —R^(a) are independently of each other        selected from the group consisting of —H, methyl, ethyl, propyl,        butyl, pentyl and hexyl.

The term “hyaluronic acid-based comprising at least X % hyaluronic acid”is used accordingly.

The term “substituted” as used herein means that one or more —H atom(s)of a molecule or moiety are replaced by a different atom or a group ofatoms, which are referred to as “substituent”.

Preferably, the one or more further optional substituents areindependently of each other selected from the group consisting ofhalogen, —CN, —COOR^(x1), —OR^(x1), —C(O)R^(x1), —C(O)N(R^(x1)R^(x1a)),—S(O)₂N(R^(x1)R^(x1a)), —S(O)N(R^(x1)R^(x1a)), —S(O)₂R^(x1),—S(O)R^(x1), —N(R^(x1))S(O)₂N(R^(x1a)R^(x1b)), —SR^(x1),—N(R^(x1)R^(x1a)), —NO₂, —OC(O)R^(x1), —N(R^(x1))C(O)R^(x1a),—N(R^(x1))S(O)₂R^(x1a), —N(R^(x1))S(O)R^(x1a), —N(R^(x1))C(O)OR^(x1a),—N(R^(x1))C(O)N(R^(x1a)R^(x1b)), —OC(O)N(R^(x1)R^(x1a)), -T⁰, C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein -T⁰, C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl are optionally substituted with one or more—R^(x2), which are the same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl are optionally interrupted by one or moregroups selected from the group consisting of -T⁰-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(x3))—, —S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—, —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a))—, —S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

—R^(x1), —R^(x1a), —R^(x1b) are independently of each other selectedfrom the group consisting of —H, -T⁰, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, andC₂₋₅₀ alkynyl; wherein -T⁰, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally substituted with one or more —R^(x2), which arethe same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T⁰-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(x3))—,—S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—; —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a))—, —S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

each T⁰ is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; whereineach T⁰ is independently optionally substituted with one or more —R^(x2)which are the same or different;

each —R^(x2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(x4), —OR^(x4), —C(O)R^(x4),—C(O)N(R^(x4)R^(x4a)), —S(O)₂N(R^(x4)R^(x4a)), —S(O)N(R^(x4)R^(x4a)),—S(O)₂R^(x4), —S(O)R^(x4), —N(R^(x4))S(O)₂N(R^(x4a)R^(x4b)), —SR^(x4),—N(R^(x4)R^(x4a)), —NO₂, —OC(O)R^(x4), —N(R^(x4)) C(O)R^(x4a),—N(R^(x4))S(O)₂R^(x4a), —N(R^(x4))S(O)R^(x4a), —N(R^(x4))C(O)OR^(x4a),—N(R^(x4))C(O)N(R^(x4a)R^(x4b)), —OC(O)N(R^(x4)R^(x4a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different;

each —R^(x3), R^(x3a), —R^(x4), —R^(x4a), —R^(x4b) is independentlyselected from the group consisting of —H and C₁₋₆ alkyl; wherein C₁₋₆alkyl is optionally substituted with one or more halogen, which are thesame or different.

More preferably, the one or more further optional substituents areindependently of each other selected from the group consisting ofhalogen, —CN, —COOR^(x1), —OR^(x1), —C(O)R^(x1), —C(O)N(R^(x1)R^(x1a)),—S(O)₂N(R^(x1)R^(x1a)), —S(O)N(R^(x1)R^(x1a)), —S(O)₂R^(x1),—S(O)R^(x1), —N(R^(x1))S(O)₂N(R^(x1a)R^(x1b)), —SR^(x1),—N(R^(x1)R^(x1a)), —NO₂, —OC(O)R^(x1), —N(R^(x1))C(O)R^(x1a),—N(R^(x1))S(O)₂R^(x1a), —N(R^(x1))S(O)R^(x1a), —N(R^(x1))C(O)OR^(x1a),—N(R^(x1))C(O)N(R^(x1a)R^(x1b)), —OC(O)N(R^(x1)R^(x1a)), -T⁰, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl; wherein -T⁰, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl are optionally substituted with one or more—R^(x2), which are the same or different and wherein C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, and C₂₋₁₀ alkynyl are optionally interrupted by one or moregroups selected from the group consisting of -T⁰-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(x3))—, —S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—, —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a)), —S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

each —R^(x1), —R^(x1a), —R^(x1b), —R^(x3), —R^(x3a) is independentlyselected from the group consisting of —H, halogen, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl;

each T⁰ is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; whereineach T⁰ is independently optionally substituted with one or more —R^(x2)which are the same or different;

each —R^(x2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(x4), —OR^(x4), —C(O)R^(x4),—C(O)N(R^(x4)R^(x4a)), —S(O)₂N(R^(x4)R^(x4a)), —S(O)N(R^(x4)R^(x4a)),—S(O)₂R^(x4), —S(O)R^(x4), —N(R^(x4))S(O)₂N(R^(x4a)R^(x4b)), —SR^(x4),—N(R^(x4)R^(x4a)), —NO₂, —OC(O)R^(x4), —N(R^(x4))C(O)R^(x4a),—N(R^(x4))S(O)₂R^(x4a), —N(R^(x4))S(O)R^(x4a), —N(R^(x4))C(O)OR^(x4a),—N(R^(x4))C(O)N(R^(x4a)R^(x4b)), —OC(O)N(R^(x4)R^(x4a)) and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different;

each —R^(x4), —R^(x4a), —R^(x4b) is independently selected from thegroup consisting of —H, halogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl;

Even more preferably, the one or more further optional substituents areindependently of each other selected from the group consisting ofhalogen, —CN, —COOR^(x1), —OR^(x1), —C(O)R^(x1), —C(O)N(R^(x1)R^(x1a)),—S(O)₂N(R^(x1)R^(x1a)), —S(O)N(R^(x1)R^(x1a)), —S(O)₂R^(x1),—S(O)R^(x1), —N(R^(x1))S(O)₂N(R^(x1a)R^(x1b)), —SR^(x1),—N(R^(x1)R^(x1a)), —NO₂, —OC(O)R^(x1), —N(R^(x1))C(O)R^(x1a),—N(R^(x1))S(O)₂R^(x1a), —N(R^(x1))S(O)R^(x1a), —N(R^(x1))C(O)OR^(x1a),—N(R^(x1))C(O)N(R^(x1a)R^(x1b), —OC(O)N(R^(x1)R^(x1a)), -T⁰, C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl; wherein -T⁰, C₁₋₆ alkyl, C₂₋₆ alkenyl,and C₂₋₆ alkynyl are optionally substituted with one or more —R^(x2),which are the same or different and wherein C₁₋₆ alkyl, C₂₋₆ alkenyl,and C₂₋₆ alkynyl are optionally interrupted by one or more groupsselected from the group consisting of -T⁰-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(x3))—, —S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—, —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a))—, —S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—; each —R^(x1), —R^(x1a),—R^(x1b), —R^(x2), —R^(x3), —R^(x3a) is independently selected from thegroup consisting of —H, halogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl;

each T⁰ is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; whereineach T⁰ is independently optionally substituted with one or more —R^(x2)which are the same or different.

Preferably, a maximum of 6 —H atoms of an optionally substitutedmolecule are independently replaced by a substituent, e.g. 5 —H atomsare independently replaced by a substituent, 4 —H atoms areindependently replaced by a substituent, 3 —H atoms are independentlyreplaced by a substituent, 2 —H atoms are independently replaced by asubstituent, or 1 —H atom is replaced by a substituent.

The term “interrupted” means that a moiety is inserted between twocarbon atoms or—if the insertion is at one of the moiety's ends—betweena carbon or heteroatom and a hydrogen atom, preferably between a carbonand a hydrogen atom.

The term “spacer” refers to any moiety that is suitable to connect twomoieties. Preferably, a spacer is selected from the group consisting of-T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y1))—, —S(O)₂N(R^(y1))—,—S(O)N(R^(y1))—, —S(O)₂—, —S(O)—, —N(R^(y1))S(O)₂N(R^(y1a))—, —S—,—N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—, —N(R^(y1))C(O)N(R^(y1a))—,—OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein-T-, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more —R^(y2), which are the same or differentand wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of-T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—, —S(O)₂N(R^(y3))—,—S(O)N(R^(y3))—, —S(O)₂—, —S(O)—, —N(R^(y3))S(O)₂N(R^(y3a))—, —S—,—N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—, —N(R^(y3))C(O)N(R^(y3a))—, and—OC(O)N(R^(y3))—;

—R^(y1) and —R^(y1a) are independently of each other selected from thegroup consisting of —H, -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl; wherein -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl areoptionally substituted with one or more —R^(y2), which are the same ordifferent, and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl areoptionally interrupted by one or more groups selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y4))—,—S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—, —S(O)₂—, —S(O)—,—N(R^(y4))S(O)₂N(R^(y4a))—, —S—, —N(R^(y4))—, —OC(OR^(y4))(R^(y4a))—,—N(R⁴)C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R^(y2), which are the same or different;

each —R^(y2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(y5), —OR^(y5), —C(O)R^(y5),—C(O)N(R^(y5)R^(y5a)), —S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)),—S(O)₂R^(y5), —S(O)R^(y5), —N(R^(y5))S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(y5)R^(y5a)), —NO₂, —OC(O)R^(y5), —N(R^(y5))C(O)R^(y5a),—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5a),—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R^(y5), —R^(y5a) and—R^(y5b) is independently selected from the group consisting of —H, andC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different.

Even more preferably the spacer is selected from -T-, —C(O)O—, —O—,—C(O)—, —C(O)N(R^(y1))—, —S(O)₂N(R^(y1))—, —S(O)N(R^(y1))—, —S(O)₂—,—S(O)—, —N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—,—OC(OR^(y1))(R^(y1a))—, —N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—,C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₂₀ alkyl,C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl are optionally substituted with one ormore —R², which are the same or different and wherein C₁₋₂₀ alkyl, C₂₋₂₀alkenyl, and C₂₋₂₀ alkynyl are optionally interrupted by one or moregroups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(y3))—, —S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R^(y3))—;

—R^(y1) and —R^(y1a) are independently of each other selected from thegroup consisting of —H, -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀alkynyl; wherein -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl areoptionally substituted with one or more —R², which are the same ordifferent, and wherein C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl areoptionally interrupted by one or more groups selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y4))—,—S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—, —S(O)₂—, —S(O)—,—N(R^(y4))S(O)₂N(R^(y4a))—, —S—, —N(R^(y4))—, —OC(OR^(y4))(R^(y4a))—,—N(R^(y4))C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R^(y2), which are the same or different;

—R^(y2) is selected from the group consisting of halogen, —CN, oxo (═O),—COOR^(Y5), —OR^(Y5), —C(O)R^(y5), —C(O)N(R^(y5)R^(y5a)),—S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)), —S(O)₂R^(y5),—S(O)R^(y5), —N(R^(y5))S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(y5)R^(y5a)), —NO₂, —OC(O)R^(y5), —N(R^(y5))C(O)R^(y5a),—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5a),—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R^(y5), —R^(Y5a) and—R^(y5b) is independently of each other selected from the groupconsisting of —H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

Even more preferably the spacer is selected from the group consisting of-T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y1))—, —S(O)₂N(R^(y1))—,—S(O)N(R^(y1))—, —S(O)₂—, —S(O)—, —N(R^(y1))S(O)₂N(R^(y1a))—, —S—,—N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—, —N(R^(y1))C(O)N(R^(y1a))—,—OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein-T-, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more —R^(y2), which are the same or differentand wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of-T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—, —S(O)₂N(R^(y3))—,—S(O)N(R^(y3))—, —S(O)₂—, —S(O)—, —N(R^(y3))S(O)₂N(R^(y3a))—, —S—,—N(R^(y3))—, —OC(OR³)(R^(y3a))—, —N(R³)C(O)N(R^(y3a))—, and—OC(O)N(R^(y3))—;

—R^(y3) and —R^(y3a) are independently selected from the groupconsisting of —H, -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;

each —R^(y2) is independently selected from the group consisting ofhalogen, and C₁₋₆ alkyl; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R^(y5), —R^(Y5a) and—R^(y5b) is independently of each other selected from the groupconsisting of —H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

As used herein, the term “C₁₋₄ alkyl” alone or in combination means astraight-chain or branched alkyl moiety having 1 to 4 carbon atoms. Ifpresent at the end of a molecule, examples of straight-chain or branchedC₁₋₄ alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl and tert-butyl. When two moieties of a molecule are linked bythe C₁₋₄ alkyl, then examples for such C₁₋₄ alkyl groups are —CH₂—,—CH—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—, —C(CH₃)₂—. Each hydrogenof a C₁₋₄ alkyl carbon may optionally be replaced by a substituent asdefined above. Optionally, a C₁₋₄ alkyl may be interrupted by one ormore moieties as defined below.

As used herein, the term “C₁₋₆ alkyl” alone or in combination means astraight-chain or branched alkyl moiety having 1 to 6 carbon atoms. Ifpresent at the end of a molecule, examples of straight-chain andbranched C₁₋₆ alkyl groups are methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl,2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. When twomoieties of a molecule are linked by the C₁₋₆ alkyl group, then examplesfor such C₁₋₆ alkyl groups are —CH₂—, —CH₂—CH₂—, —CH(CH₃)—,—CH₂—CH₂—CH₂—, —CH(C₂H₅)— and —C(CH₃)₂—. Each hydrogen atom of a C₁₋₆carbon may optionally be replaced by a substituent as defined above.Optionally, a C₁₋₆ alkyl may be interrupted by one or more moieties asdefined below.

Accordingly, “C₁₋₁₀ alkyl”, “C₁₋₂₀ alkyl” or “C₁₋₅₀ alkyl” means analkyl chain having 1 to 10, 1 to 20 or 1 to 50 carbon atoms,respectively, wherein each hydrogen atom of the C₁₋₁₀, C₁₋₂₀ or C₁₋₅₀carbon may optionally be replaced by a substituent as defined above.Optionally, a C₁₋₁₀ or C₁₋₅₀ alkyl may be interrupted by one or moremoieties as defined below.

As used herein, the term “C₂₋₆ alkenyl” alone or in combination means astraight-chain or branched hydrocarbon moiety comprising at least onecarbon-carbon double bond having 2 to 6 carbon atoms. If present at theend of a molecule, examples are —CH═CH₂, —CH═CH—CH₃, —CH₂—CH═CH₂,—CH═CHCH₂—CH₃ and —CH═CH—CH═CH₂. When two moieties of a molecule arelinked by the C₂₋₆ alkenyl group, then an example for such C₂₋₆ alkenylis —CH═CH—. Each hydrogen atom of a C₂₋₆ alkenyl moiety may optionallybe replaced by a substituent as defined above. Optionally, a C₂₋₆alkenyl may be interrupted by one or more moieties as defined below.

Accordingly, the term “C₂₋₁₀ alkenyl”, “C₂₋₂₀ alkenyl” or “C₂₋₅₀alkenyl” alone or in combination means a straight-chain or branchedhydrocarbon moiety comprising at least one carbon-carbon double bondhaving 2 to 10, 2 to 20 or 2 to 50 carbon atoms. Each hydrogen atom of aC₂₋₁₀ alkenyl, C₂₋₂₀ alkenyl or C₂₋₅₀ alkenyl group may optionally bereplaced by a substituent as defined above. Optionally, a C₂₋₁₀ alkenyl,C₂₋₂₀ alkenyl or C₂₋₅₀ alkenyl may be interrupted by one or moremoieties as defined below.

As used herein, the term “C₂₋₆ alkynyl” alone or in combination meansstraight-chain or branched hydrocarbon moiety comprising at least onecarbon-carbon triple bond having 2 to 6 carbon atoms. If present at theend of a molecule, examples are —C≡CH, —CH₂—C≡CH, CH₂—CH₂—C≡CH andCH₂—C≡C—CH₃. When two moieties of a molecule are linked by the alkynylgroup, then an example is —C≡C—. Each hydrogen atom of a C₂₋₆ alkynylgroup may optionally be replaced by a substituent as defined above.Optionally, one or more double bond(s) may occur. Optionally, a C₂₋₆alkynyl may be interrupted by one or more moieties as defined below.

Accordingly, as used herein, the term “C₂₋₁₀ alkynyl”, “C₂₋₂₀ alkynyl”and “C₂₋₅₀ alkynyl” alone or in combination means a straight-chain orbranched hydrocarbon moiety comprising at least one carbon-carbon triplebond having 2 to 10, 2 to 20 or 2 to 50 carbon atoms, respectively. Eachhydrogen atom of a C₂₋₁₀ alkynyl, C₂₋₂₀ alkynyl or C₂₋₅₀ alkynyl groupmay optionally be replaced by a substituent as defined above.Optionally, one or more double bond(s) may occur. Optionally, a C₂₋₁₀alkynyl, C₂₋₂₀ alkynyl or C₂₋₅₀ alkynyl may be interrupted by one ormore moieties as defined below.

As mentioned above, a C₁₋₄ alkyl, C₁₋₆ alkyl, C₁₋₁₀ alkyl, C₁₋₂₀ alkyl,C₁₋₅₀ alkyl, C₂₋₆ alkenyl, C₂₋₁₀ alkenyl, C₂₋₂₀ alkenyl, C₂₋₅₀ alkenyl,C₂₋₆ alkynyl, C₂₋₁₀ alkynyl, C₂₋₂₀ alkenyl or C₂₋₅₀ alkynyl mayoptionally be interrupted by one or more moieties which are preferablyselected from the group consisting of

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent; and —R and —R^(a) are independently of each other        selected from the group consisting of —H, methyl, ethyl, propyl,        butyl, pentyl and hexyl.

As used herein, the term “C₃₋₁₀ cycloalkyl” means a cyclic alkyl chainhaving 3 to 10 carbon atoms, which may be saturated or unsaturated, e.g.cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. Each hydrogen atom ofa C₃₋₁₀ cycloalkyl carbon may be replaced by a substituent as definedabove. The term “C₃₋₁₀ cycloalkyl” also includes bridged bicycles likenorbornane or norbornene.

The term “8- to 30-membered carbopolycyclyl” or “8- to 30-memberedcarbopolycycle” means a cyclic moiety of two or more rings with 8 to 30ring atoms, where two neighboring rings share at least one ring atom andthat may contain up to the maximum number of double bonds (aromatic ornon-aromatic ring which is fully, partially or un-saturated). Preferablya 8-to 30-membered carbopolycyclyl means a cyclic moiety of two, three,four or five rings, more preferably of two, three or four rings.

As used herein, the term “3- to 10-membered heterocyclyl” or “3- to10-membered heterocycle” means a ring with 3, 4, 5, 6, 7, 8, 9 or 10ring atoms that may contain up to the maximum number of double bonds(aromatic or non-aromatic ring which is fully, partially orun-saturated) wherein at least one ring atom up to 4 ring atoms arereplaced by a heteroatom selected from the group consisting of sulfur(including —S(O)—, —S(O)₂—), oxygen and nitrogen (including ═N(O)—) andwherein the ring is linked to the rest of the molecule via a carbon ornitrogen atom. Examples for 3- to 10-membered heterocycles include butare not limited to aziridine, oxirane, thiirane, azirine, oxirene,thiirene, azetidine, oxetane, thietane, furan, thiophene, pyrrole,pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole,oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine and homopiperazine. Each hydrogen atomof a 3- to 10-membered heterocyclyl or 3-to 10-membered heterocyclicgroup may be replaced by a substituent as defined below.

As used herein, the term “8- to 11-membered heterobicyclyl” or “8- to11-membered heterobicycle” means a heterocyclic moiety of two rings with8 to 11 ring atoms, where at least one ring atom is shared by both ringsand that may contain up to the maximum number of double bonds (aromaticor non-aromatic ring which is fully, partially or un-saturated) whereinat least one ring atom up to 6 ring atoms are replaced by a heteroatomselected from the group consisting of sulfur (including —S(O)—,—S(O)₂—), oxygen and nitrogen (including ═N(O)—) and wherein the ring islinked to the rest of the molecule via a carbon or nitrogen atom.Examples for an 8- to 11-membered heterobicycle are indole, indoline,benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole,benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine andpteridine. The term 8-to 11-membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of an8- to 11-membered heterobicyclyl or 8- to 11-membered heterobicyclecarbon may be replaced by a substituent as defined below.

Similarly, the term “8- to 30-membered heteropolycyclyl” or “8- to30-membered heteropolycycle” means a heterocyclic moiety of more thantwo rings with 8 to 30 ring atoms, preferably of three, four or fiverings, where two neighboring rings share at least one ring atom and thatmay contain up to the maximum number of double bonds (aromatic ornon-aromatic ring which is fully, partially or unsaturated), wherein atleast one ring atom up to 10 ring atoms are replaced by a heteroatomselected from the group of sulfur (including —S(O)—, —S(O)₂—), oxygenand nitrogen (including=N(O)—) and wherein the ring is linked to therest of a molecule via a carbon or nitrogen atom.

It is understood that the phrase “the pair R^(x)/R^(y) is joinedtogether with the atom to which they are attached to form a C₃₋₁₀cycloalkyl or a 3- to 10-membered heterocyclyl” in relation with amoiety of the structure

means that Rx and Ry form the following structure:

wherein R is C₃₋₁₀ cycloalkyl or 3- to 10-membered heterocyclyl.

It is also understood that the phrase “the pair R/R^(y) is jointtogether with the atoms to which they are attached to form a ring A” inrelation with a moiety of the structure

means that R^(x) and R form the following structure:

As used herein, the term “terminal alkyne” means a moiety

As used herein, “halogen” means fluoro, chloro, bromo or iodo. It isgenerally preferred that halogen is fluoro or chloro.

In general, the term “comprise” or “comprising” also encompasses“consist of” or “consisting of”.

It is understood that in formula (Ia) and (Ib) -D is connected to -L¹-via a covalent and reversible linkage.

In another aspect the present invention relates to a PTH prodrug or apharmaceutically acceptable salt thereof comprising a conjugate D-L,wherein

-   -   -D is a PTH moiety; and    -   -L comprises a reversible prodrug linker moiety -L¹-, which        moiety -L¹- is connected to the PTH moiety -D through a        functional group of PTH;    -   wherein -L¹- is substituted with -L²-Z′ and is optionally        further substituted; wherein    -   -L²- is a single chemical bond or a spacer moiety; and    -   -Z′ is a water-insoluble carrier moiety.

It is understood that a multitude of moieties -L²-L¹-D is connected to awater-insoluble carrier -Z′ and that the linkage between -D and -L¹- iscovalent and reversible.

Preferably, -D has the sequence of SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ IDNO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 orSEQ ID NO:115. More preferably -D has the sequence of SEQ ID NO:50, SEQID NO:51, SEQ ID NO:52, SEQ ID NO:110, SEQ ID NO:111 or SEQ ID NO:112.

In one embodiment -D has the sequence of SEQ ID NO:50.

In another embodiment -D has the sequence of SEQ ID NO:52.

In another embodiment -D has the sequence of SEQ ID NO:110.

In another embodiment -D has the sequence of SEQ ID NO:111.

In another embodiment -D has the sequence of SEQ ID NO:112.

Most preferably -D has the sequence of SEQ ID NO:51.

The moiety -L¹- is either conjugated to a functional group of the sidechain of an amino acid residue of -D, to the N-terminal amine functionalgroup or to the C-terminal carboxyl functional group of -D or to anitrogen atom in the backbone polypeptide chain of -D. Attachment toeither the N-terminus or C-terminus can either be directly through thecorresponding amine or carboxyl functional group, respectively, orindirectly wherein a spacer moiety is first conjugated to the amine orcarboxyl functional group to which spacer moiety -L¹- is conjugated.

Preferably, the amino acid residue of PTH to which -L¹- is conjugatedcomprises a functional group selected from the group consistingcarboxylic acid, primary and secondary amine, maleimide, thiol, sulfonicacid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine,isocyanate, isothiocyanate, phosphoric acid, phosphonic acid,haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine,sulfate, disulfide, vinyl sulfone, vinyl ketone, diazoalkane, oxirane,guanidine and aziridine. Even more preferably the amino acid residue ofPTH to which -L¹- is conjugated comprises a functional group selectedfrom the group consisting hydroxyl, primary and secondary amine andguanidine. Even more preferably the amino acid residue of PTH to which-L¹- is conjugated comprises a primary or secondary amine functionalgroup. Most preferably the amino acid residue of PTH to which -L¹- isconjugated comprises a primary amine functional group.

If the moiety -L¹- is conjugated to a functional group of the side chainof an amino acid residue of PTH said amino acid residue is selected fromthe group consisting of proteinogenic amino acid residues andnon-proteinogenic amino acid residues.

In one embodiment -L¹- is conjugated to a functional group of the sidechain of a non-proteinogenic amino acid residue of PTH. It is understoodthat such non-proteinogenic amino acid is not found in the sequence ofnative PTH or fragments thereof and that it may only be present invariants, analogs, orthologs, homologs and derivatives of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a proteinogenic amino acid residue of PTH. Preferably,said amino acid is selected from the group consisting of histidine,lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamicacid and arginine. Even more preferably said amino acid is selected fromthe group consisting of lysine, aspartic acid, arginine and serine. Evenmore preferably said amino acid is selected from the group consisting oflysine, arginine and serine.

In one embodiment -L¹- is conjugated to a functional group of the sidechain of a histidine of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a lysine of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a tryptophan of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a serine of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a threonine of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a tyrosine of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a aspartic acid of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of a glutamic acid of PTH.

In another embodiment -L¹- is conjugated to a functional group of theside chain of an arginine of PTH.

It is understood that not every PTH moiety may comprise all of theseamino acid residues.

In a preferred embodiment -L¹- is conjugated to the N-terminal aminefunctional group of PTH, either directly through the corresponding aminefunctional group or indirectly wherein a spacer moiety is firstconjugated to the amine functional group to which spacer moiety -L¹- isconjugated. Even more preferably, -L¹- is directly conjugated to theN-terminal amine functional group of PTH, preferably PTH 1-34, i.e. PTHhaving the sequence of SEQ ID NO:51.

It was surprisingly found that N-terminal attachment of -L¹- isadvantageous, i.e. attachment of -L¹- to the N-terminus of PTH, becauseit was found that such attachment site protects the N-terminus which iscrucial for PTH activity. Furthermore, it was surprisingly found thatthe main metabolite formed from a PTH prodrug with N-terminal attachmentof -L¹- is PTH 1-33, i.e. the 33 N-terminal amino acids of PTH, whichmetabolite is known to be active.

In another embodiment -L¹- is conjugated to the C-terminal functionalgroup of PTH, either directly through the corresponding carboxylfunctional group or indirectly wherein a spacer moiety is firstconjugated to the carboxyl functional group to which spacer moiety -L¹-is conjugated.

Most preferably L¹- is directly conjugated to the N-terminal aminefunctional group of PTH.

The moiety -L¹- can be connected to -D through any type of linkage,provided that it is reversible. Preferably, -L¹- is connected to -Dthrough a linkage selected from the group consisting of amide, ester,carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide andacylguanidine. Even more preferably -L¹- is connected to -D through alinkage selected from the group consisting of amide, ester, carbamateand acylguanidin. It is understood that some of these linkages per seare not reversible, but that in the present invention neighboring groupscomprised in -L¹- render these linkage reversible.

In one embodiment -L¹- is connected to -D through an ester linkage.

In another embodiment -L¹- is connected to -D through a carbamatelinkage.

In another embodiment -L¹- is connected to -D through an acylguanidine.

In a preferred embodiment -L¹- is connected to -D through an amidelinkage.

The moiety -L¹- is a reversible prodrug linker from which the drug, i.e.PTH, is released in its free form, i.e. it is a traceless prodruglinker. Suitable prodrug linkers are known in the art, such as forexample the reversible prodrug linker moieties disclosed in WO2005/099768 A2, WO 2006/136586 A2, WO 2011/089216 A1 and WO 2013/024053A1, which are incorporated by reference herewith.

In another embodiment -L¹- is a reversible prodrug linker as describedin WO 2011/012722 A1, WO 2011/089214 A1, WO 2011/089215 A1, WO2013/024052 A1 and WO 2013/160340 A1 which are incorporated by referenceherewith.

A particularly preferred moiety -L¹- is disclosed in WO 2009/095479 A2.Accordingly, in a preferred embodiment the moiety -L¹- is of formula(II):

-   -   wherein the dashed line indicates the attachment to a nitrogen,        hydroxyl or thiol of -D which is a PTH moiety;    -   —X— is —C(R⁴R^(4a))—; —N(R⁴)—; —O—; —C(R⁴R^(4a))—C(R⁵R^(5a))—;        —C(R⁵R^(5a))—C(R⁴R^(4a))—; —C(R⁴R^(4a))—N(R⁶)—;        —N(R⁶)—C(R⁴R^(4a))—; —C(R⁴R^(4a))—O—; —O—C(R⁴R^(4a))—; or        —C(R⁷R^(7a))—;    -   X¹ is C; or S(O);    -   —X²— is —C(R⁸R^(8a))—; or —C(R⁸R^(8a))—C(R⁹R^(9a))—;    -   ═X³ is ═O; ═S; or ═N—CN;    -   —R¹, —R^(1a), —R², —R^(2a), —R⁴, —R^(4a), —R⁵, —R^(5a), —R⁶,        —R⁸, —R^(8a), —R⁹, —R^(9a) are independently selected from the        group consisting of —H; and C₁₋₆ alkyl;    -   —R³, —R^(3a) are independently selected from the group        consisting of —H; and C₁₋₆ alkyl, provided that in case one of        —R³, —R^(3a) or both are other than —H they are connected to N        to which they are attached through an SP³-hybridized carbon        atom;    -   —R⁷ is —N(R¹⁰, R^(10a)); or —NR¹⁰—(C═O)—R¹¹;    -   —R^(7a)—, —R¹⁰, —R^(10a), —R¹¹ are independently of each other        —H; or C₁₋₆ alkyl;    -   optionally, one or more of the pairs —R^(1a)/—R^(4a),        —R^(1a)/—R^(5a), —R^(1a)/—R^(7a), —R^(4a)/—R^(5a),        —R^(8a)/—R^(9a) form a chemical bond;    -   optionally, one or more of the pairs —R¹/—R^(1a), —R²/—R^(2a),        —R⁴/—R^(4a), —R⁵/—R^(5a), —R⁸/—R^(8a),    -   —R⁹/—R^(9a) are joined together with the atom to which they are        attached to form a C₃₋₁₀ cycloalkyl; or 3- to 10-membered        heterocyclyl;    -   optionally, one or more of the pairs —R¹/—R⁴, —R¹/—R⁵, —R¹/—R⁶,        —R¹/—R^(7a), —R⁴/—R⁵, —R⁴/—R⁶, —R⁸/—R⁹, —R²/—R³ are joined        together with the atoms to which they are attached to form a        ring A;    -   optionally, R³/R^(3a) are joined together with the nitrogen atom        to which they are attached to form a 3- to 10-membered        heterocycle;    -   A is selected from the group consisting of phenyl; naphthyl;        indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 3- to        10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl;        and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (II) is not        replaced by -L²-Z or -L²-Z′ or a substituent;    -   wherein    -   -L²- is a single chemical bond or a spacer;    -   —Z is a water-soluble carrier; and    -   Z′ is a water-insoluble carrier.

Preferably -L¹- of formula (II) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (II) is not further substituted.

It is understood that if —R³/—R^(3a) of formula (II) are joined togetherwith the nitrogen atom to which they are attached to form a 3- to10-membered heterocycle, only such 3- to 10-membered heterocycles may beformed in which the atoms directly attached to the nitrogen areSP³-hybridized carbon atoms. In other words, such 3- to 10-memberedheterocycle formed by —R³/—R^(3a) together with the nitrogen atom towhich they are attached has the following structure:

-   -   wherein    -   the dashed line indicates attachment to the rest of -L¹-;    -   the ring comprises 3 to 10 atoms comprising at least one        nitrogen; and    -   R^(#) and R^(##) represent an SP³-hydridized carbon atom.

It is also understood that the 3- to 10-membered heterocycle may befurther substituted.

Exemplary embodiments of suitable 3- to 10-membered heterocycles formedby —R³/—R^(3a) of formula (II) together with the nitrogen atom to whichthey are attached are the following:

-   -   wherein    -   dashed lines indicate attachment to the rest of the molecule;        and    -   —R is selected from the group consisting of —H and C₁₋₆ alkyl.

-L¹- of formula (II) may optionally be further substituted. In general,any substituent may be used as far as the cleavage principle is notaffected, i.e. the hydrogen marked with the asterisk in formula (II) isnot replaced and the nitrogen of the moiety

of formula (II) remains part of a primary, secondary or tertiary amine,i.e. —R³ and —R^(3a) are independently of each other —H or are connectedto —N< through an SP³-hybridized carbon atom.

In one embodiment —R¹ or —R^(1a) of formula (II) is substituted with-L²-Z or -L²-Z′. In another embodiment —R² or —R^(2a) of formula (II) issubstituted with -L²-Z or -L²-Z′. In another embodiment —R³ or —R^(3a)of formula (II) is substituted with -L²-Z or -L²-Z′. In anotherembodiment —R⁴ of formula (II) is substituted with -L²-Z or -L²-Z′. Inanother embodiment —R⁵ or —R^(5a) of formula (II) is substituted with-L²-Z or -L²-Z′. In another embodiment —R⁶ of formula (II) issubstituted with -L²-Z or -L²-Z′. In another embodiment —R⁷ or —R^(7a)of formula (II) is substituted with -L²-Z or -L²-Z′. In anotherembodiment —R⁸ or —R^(8a) of formula (II) is substituted with -L²-Z or-L²-Z′. In another embodiment —R⁹ or —R^(9a) of formula (II) issubstituted with -L²-Z or -L²-Z′. In another embodiment —R¹⁰ issubstituted with -L²-Z or -L²-Z′. In another embodiment —R¹¹ issubstituted with -L²-Z or -L²-Z′.

Preferably, —X— of formula (II) is selected from the group consisting of—C(R⁴R^(4a))—, —N(R⁴)— and —C(R⁷R^(7a))—.

In one embodiment —X— of formula (II) is —C(R⁴R^(4a))—.

In one preferred embodiment —X— of formula (II) is —C(R⁷R^(7a))—.

Preferably, —R⁷ of formula (II) is —NR¹⁰—(C═O)—R¹¹.

Preferably, —R^(7a) of formula (II) is selected from —H, methyl andethyl. Most preferably —R^(7a) of formula (II) is —H.

Preferably, —R¹⁰ is selected from —H, methyl and ethyl. Most preferably—R¹⁰ is methyl.

Preferably, —R¹¹ is selected from —H, methyl and ethyl. Most preferably—R¹¹ is —H.

Preferably, —R¹¹ is substituted with -L²-Z or -L²-Z′.

In another preferred embodiment —X— of formula (II) is —N(R⁴)—.

Preferably, —R⁴ is selected from the group consisting of —H, methyl andethyl. Preferably, —R⁴ is —H.

Preferably, X¹ of formula (II) is C.

Preferably, ═X³ of formula (II) is ═O.

Preferably, —X²— of formula (II) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (II) is —H. Even more preferablyboth —R⁸ and —R^(8a) of formula (II) are —H.

Preferably, —R and —R^(1a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl.

In one preferred embodiment at least one of —R¹ and —R^(1a) of formula(II) is —H, more preferably both —R¹ and —R^(1a) of formula (II) are —H.

In another preferred embodiment at least one of —R¹ and —R^(1a) offormula (II) is methyl, more preferably both —R¹ and —R^(1a) of formula(II) are methyl.

Preferably, —R² and —R^(2a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R² and —R^(2a) of formula (II) is —H.

Even more preferably both —R² and —R^(2a) of formula (II) are H.

Preferably, —R³ and —R^(3a) of formula (II) are independently selectedfrom the group consisting of —H, methyl, ethyl, propyl and butyl.

In one preferred embodiment at least one of —R³ and —R^(3a) of formula(II) is methyl, more preferably —R³ of formula (II) is methyl and—R^(3a) of formula (II) is —H.

In another preferred embodiment —R³ and —R^(3a) of formula (II) are both—H.

Preferably, -D is connected to -L¹- through a nitrogen by forming anamide bond.

In one preferred embodiment the moiety -L¹- is of formula (IIa-i):

-   -   wherein    -   the dashed line indicates the attachment to a nitrogen of -D        which is a PTH moiety by forming an amide bond;    -   —R¹, —R^(1a), —R², —R^(2a), —R³, —R^(3a), —R⁷, —R^(7a) and —X²—        are used as defined in formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIa-i) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIa-i) is substituted with one moiety -L²-Zor -L²-Z′.

Preferably the moiety -L¹- of formula (IIa-i) is not furthersubstituted.

Preferably, —R¹ and —R^(1a) of formula (IIa-i) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably, at least one of —R¹ and —R^(1a) of formula (IIa-i) is —H.Even more preferably both —R¹ and —R^(1a) of formula (IIa-i) are —H.

Preferably, —R⁷ of formula (IIa-i) is —NR¹⁰—(C═O)—R¹¹.

Preferably, —R^(7a) of formula (II-i) is selected from —H, methyl andethyl. Most preferably —R^(7a) of formula (II-i) is —H.

Preferably, —R¹⁰ of formula (IIa-i) is selected from —H, methyl andethyl. Most preferably —R¹⁰ of formula (IIa-i) is methyl.

Preferably, —R¹¹ of formula (IIa-i) is selected from —H, methyl andethyl. Most preferably —R¹¹ of formula (IIa-i) is —H.

Preferably, —R¹¹ of formula (IIa-i) is substituted with -L²-Z or -L²-Z′.

Preferably, —X²— of formula (IIa-i) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIa-i) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (IIa-i) is —H. Even morepreferably both —R⁸ and —R^(8a) of formula (IIa-i) are —H.

Preferably, —R² and —R^(2a) of formula (IIa-i) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably, at least one of —R² and —R^(2a) of formula (IIa-i) is —H.Even more preferably both —R² and —R^(2a) of formula (IIa-i) are H.

Preferably, —R³ and —R^(3a) of formula (IIa-i) are independentlyselected from the group consisting of —H, methyl, ethyl, propyl andbutyl. Even more preferably at least one of —R³ and —R^(3a) of formula(IIa-i) is methyl.

Preferably, —R³ of formula (IIa-i) is —H and —R^(3a) of formula (IIa-i)is methyl.

More preferably the moiety -L¹- is of formula (IIa-ii):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond;    -   —R², —R^(2a), —R¹⁰, —R¹¹ and —X²— are used as defined in formula        (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIa-ii) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIa-ii) is substituted with one moiety -L²-Zor -L²-Z′.

Preferably the moiety -L¹- of formula (IIa-ii) is not furthersubstituted.

Preferably, —X²— of formula (IIa-ii) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIa-ii) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably at least one of —R⁸ and —R^(8a) of formula (IIa-ii) is —H.Even more preferably both —R⁸ and —R^(8a) of formula (IIa-ii) are —H.

Preferably, —R³ and —R^(3a) of formula (IIa-ii) are independentlyselected from the group consisting of —H, methyl, ethyl, propyl andbutyl. Even more preferably at least one of —R³ and —R^(3a) of formula(IIa-ii) is methyl.

Preferably, —R³ of formula (IIa-ii) is —H and —R^(3a) of formula(IIa-ii) is methyl.

Preferably, —R¹⁰ of formula (IIa-ii) is selected from —H, methyl andethyl. Most preferably —R¹⁰ of formula (IIa-ii) is methyl.

Preferably, —R¹¹ of formula (IIa-ii) is selected from —H, methyl andethyl. Most preferably —R¹¹ of formula (IIa-ii) is —H.

Preferably, —R¹¹ of formula (IIa-ii) is substituted with -L²-Z or-L²-Z′.

In an even more preferred embodiment the moiety -L¹- is of formula(IIa-ii′):

-   -   wherein    -   wherein the dashed line indicates the attachment to a nitrogen        of D which is a PTH moiety by forming an amide bond;    -   the dashed line marked with the asterisk indicates attachment to        -L²-;    -   —R³, —R^(3a), —R¹⁰ and —X²— are used as defined in formula (II);        and    -   wherein -L¹- is optionally further substituted, provided that        the hydrogen marked with the asterisk in formula (IIa-ii′) is        not replaced by a substituent.

Preferably the moiety -L¹- of formula (IIa-ii′) is not furthersubstituted.

Preferably, —X²— of formula (IIa-ii′) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIa-ii′) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably at least one of —R⁸ and —R^(8a) of formula (IIa-ii′) is —H.Even more preferably both —R⁸ and —R^(8a) of formula (IIa-ii′) are —H.

Preferably, —R³ and —R^(3a) of formula (IIa-ii′) are independentlyselected from the group consisting of —H, methyl, ethyl, propyl andbutyl. Even more preferably at least one of —R³ and —R^(3a) of formula(IIa-ii′) is methyl.

Preferably, —R³ of formula (IIa-ii′) is —H and —R^(3a) of formula(IIa-ii′) is methyl.

Preferably, —R¹⁰ of formula (IIa-ii′) is selected from —H, methyl andethyl. Most preferably —R¹⁰ of formula (IIa-ii′) is methyl.

Even more preferably the moiety -L¹- is of formula (IIa-iii):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond; and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIa-iii) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIa-iii) is substituted with one moiety-L²-Z or -L²-Z′.

Preferably the moiety -L¹- of formula (IIa-iii) is not furthersubstituted.

Most preferably the moiety -L¹- is of formula (IIa-iii′):

-   -   wherein    -   wherein the dashed line indicates the attachment to a nitrogen        of D which is a PTH moiety by forming an amide bond;    -   the dashed line marked with the asterisk indicates attachment to        -L²-; —R², —R^(2a), —R³, —R^(3a) and —X²— are used as defined in        formula (II); and    -   wherein -L¹- is optionally further substituted, provided that        the hydrogen marked with the asterisk in formula (IIa-iii′) is        not replaced by a substituent.

Preferably the moiety -L¹- of formula (IIa-iii′) is not furthersubstituted.

In another preferred embodiment the moiety -L¹- is of formula (IIb-i)

-   -   wherein    -   the dashed line indicates the attachment to a nitrogen of -D        which is a PTH moiety by forming an amide bond;    -   —R¹, —R^(1a), —R², —R^(2a), —R³, —R^(3a), —R⁴ and —X²— are used        as defined in formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIb-i) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIb-i) is substituted with one moiety -L²-Zor -L²-Z′.

Preferably the moiety -L¹- of formula (IIb-i) is not furthersubstituted.

Preferably, —R¹ and —R^(1a) of formula (IIb-i) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably, at least one of —R¹ and —R^(1a) of formula (IIb-i) ismethyl. Even more preferably both —R¹ and —R^(1a) of formula (IIb-i) aremethyl.

Preferably, —R⁴ of formula (IIb-i) is selected from the group consistingof —H, methyl and ethyl. More preferably, —R⁴ of formula (IIb-i) is —H.

Preferably, —X²— of formula (IIb-i) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIb-i) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (IIb-i) is —H. Even morepreferably both —R⁸ and —R^(8a) of formula (IIb-i) are —H.

Preferably, —R² and —R^(2a) of formula (IIb-i) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably, at least one of —R² and —R^(2a) of formula (IIb-i) is —H.Even more preferably both —R² and —R^(2a) of formula (IIb-i) are H.

Preferably, —R³ and —R^(3a) of formula (IIb-i) are independentlyselected from the group consisting of —H, methyl, ethyl, propyl andbutyl. Even more preferably at least one of —R³ and —R^(3a) of formula(IIb-i) is —H. Even more preferably both —R³ and —R^(3a) of formula(IIb-i) are —H.

More preferably the moiety -L¹- is of formula (IIb-ii):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond;    -   —R², —R^(2a), —R³, —R^(3a) and —X²— are used as defined in        formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIb-ii) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIb-ii) is substituted with one moiety -L²-Zor -L²-Z′.

Preferably the moiety -L¹- of formula (IIb-ii) is not furthersubstituted.

Preferably, —X²— of formula (IIb-ii) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIb-ii) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably at least one of —R⁸ and —R^(8a) of formula (IIb-ii) is —H.Even more preferably both —R⁸ and —R^(8a) of formula (IIb-ii) are —H.

Preferably, —R² and —R^(2a) of formula (IIb-ii) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably, at least one of —R² and —R^(2a) of formula (IIb-ii) is —H.Even more preferably both —R² and —R^(2a) of formula (IIb-ii) are H.

Preferably, —R³ and —R^(3a) of formula (IIb-ii) are independentlyselected from the group consisting of —H, methyl, ethyl, propyl andbutyl. Even more preferably at least one of —R³ and —R^(3a) of formula(IIb-ii) is —H. Even more preferably both —R³ and —R^(3a) of formula(IIb-ii) are —H.

Even more preferably the moiety -L¹- is of formula (IIb-ii′):

-   -   wherein    -   the dashed line indicates the attachment to a nitrogen of -D        which is a PTH moiety by forming an amide bond;    -   —R², —R^(2a), —R³, —R^(3a) and —X²— are used as defined in        formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIb-ii′) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably the moiety -L¹- of formula (IIb-ii′) is not furthersubstituted.

Preferably, —X²— of formula (IIb-ii′) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIb-ii′) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably at least one of —R⁸ and —R^(8a) of formula (IIb-ii′) is —H.Even more preferably both —R⁸ and —R^(8a) of formula (IIb-ii′) are —H.

Preferably, —R² and —R^(2a) of formula (IIb-ii′) are independentlyselected from the group consisting of —H, methyl and ethyl. Morepreferably, at least one of —R² and —R^(2a) of formula (IIb-ii′) is —H.Even more preferably both —R² and —R^(2a) of formula (IIb-ii′) are H.

Preferably, —R³ and —R^(3a) of formula (IIb-ii′) are independentlyselected from the group consisting of —H, methyl, ethyl, propyl andbutyl. Even more preferably at least one of —R³ and —R^(3a) of formula(IIb-ii′) is —H. Even more preferably both —R³ and —R^(3a) of formula(IIb-ii′) are —H.

Even more preferably the moiety -L¹- is of formula (IIb-iii):

-   -   wherein    -   the dashed line indicates the attachment to a nitrogen of -D        which is a PTH moiety by forming an amide bond; and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIb-iii) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIb-iii) is substituted with one moiety-L²-Z or -L²-Z′.

Preferably the moiety -L¹- of formula (IIb-iii) is not furthersubstituted.

Most preferably the moiety -L¹- is of formula (IIb-iii′):

-   -   wherein    -   the dashed line indicates the attachment to a nitrogen of -D        which is a PTH moiety by forming an amide bond;    -   —R², —R^(2a), —R³, —R^(3a) and —X²— are used as defined in        formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIb-iii′) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably the moiety -L¹- of formula (IIb-iii′) is not furthersubstituted.

Another preferred moiety -L¹- is disclosed in unpublished Europeanpatent application 14180004, which corresponds to the internationalapplication with the application number PCT/EP2015/067929. Accordingly,in another preferred embodiment the moiety -L¹- is of formula (III):

-   -   wherein    -   the dashed line indicates attachment to a primary or secondary        amine or hydroxyl of -D which is a PTH moiety by forming an        amide or ester linkage, respectively;    -   —R¹, —R^(1a), —R², —R^(2a), —R³ and —R^(3a) are independently of        each other selected from the group consisting of —H,        —C(R⁸R^(8a)R^(8b)), —C(═O)R⁸, —C≡N, —C(═NR⁸)R^(8a),        —CR⁸(═CR^(8a)R^(8b)), —C≡CR⁸ and -T;    -   —R⁴, —R⁵ and —R^(5a) are independently of each other selected        from the group consisting of —H, —C(R⁹R^(9a)R^(9b)) and -T;    -   a1 and a2 are independently of each other 0 or 1;    -   each —R⁶, —R^(6a), —R⁷, —R^(7a), —R⁸, —R^(8a), —R⁸, —R⁹,        —R^(9a), —R^(9b) are independently of each other selected from        the group consisting of —H, halogen, —CN, —COOR¹⁰, —OR¹⁰,        —C(O)R¹⁰, —C(O)N(R¹⁰R^(10a)), —S(O)₂N(R¹⁰R^(10a)),        —S(O)N(R¹⁰R^(10a)), —S(O)₂R¹⁰, —S(O)R¹⁰,        —N(R¹⁰)S(O)₂N(R^(10a)R^(10b)), —SR¹⁰, —N(R¹⁰R^(10a)), —NO₂,        —OC(O)R¹⁰, —N(R¹⁰)C(O)R^(10a), —N(R¹⁰)S(O)₂R^(10a),        —N(R¹⁰)S(O)R^(10a), —N(R¹⁰)C(O)OR^(10a),        —N(R¹⁰)C(O)N(R^(10a)R^(10b)), —OC(O)N(R¹⁰R^(10a)), -T, C₁₋₂₀        alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl; wherein -T, C₁₋₂₀        alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl are optionally        substituted with one or more —R¹¹, which are the same or        different and wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀        alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R¹²)—, —S(O)₂N(R¹²)—, —S(O)N(R¹²)—, —S(O)₂—, —S(O)—,        —N(R¹²)S(O)₂N(R^(12a))—, —S—, —N(R¹²)—, —OC(OR¹²)(R^(12a))—,        —N(R¹²)C(O)N(R^(12a))—, and —OC(O)N(R¹²)—;    -   each —R¹⁰, —R^(10a), —R^(10b) is independently selected from the        group consisting of —H, -T, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and        C₂₋₂₀ alkynyl; wherein -T, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀        alkynyl are optionally substituted with one or more —R¹¹, which        are the same or different and wherein C₁₋₂₀ alkyl, C₂₋₂₀        alkenyl, and C₂₋₂₀ alkynyl are optionally interrupted by one or        more groups selected from the group consisting of -T-, —C(O)O—,        —O—, —C(O)—, —C(O)N(R)—, —S(O)₂N(R¹²)—, —S(O)N(R¹²)—, —S(O)₂—,        —S(O)—, —N(R¹²)S(O)₂N(R^(12a))—, —S—, —N(R¹²)—,        —OC(OR¹²)(R^(12a))—, —N(R¹²)C(O)N(R^(12a))—, and —OC(O)N(R¹²)—;    -   each T is independently of each other selected from the group        consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,        C₃₋₁₀ cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to        11-membered heterobicyclyl; wherein each T is independently        optionally substituted with one or more —R¹¹, which are the same        or different;    -   each —R¹¹ is independently of each other selected from halogen,        —CN, oxo (═O), —COOR¹³, —OR¹³, —C(O)R¹³, —C(O)N(R¹³R^(13a)),        —S(O)₂N(R¹³R^(13a)), —S(O)N(R¹³R^(13a)), —S(O)₂R¹³, —S(O)R¹³,        —N(R¹³)S(O)₂N(R^(13a)R^(13b)), —SR¹³, —N(R¹³R^(13a)), —NO₂,        —OC(O)R¹³, —N(R¹³)C(O)R^(13a), —N(R¹³)S(O)₂R^(13a),        —N(R¹³)S(O)R^(13a), —N(R¹³)C(O)OR¹³a,        —N(R¹³)C(O)N(R^(13a)R^(13b)), —OC(O)N(R¹³R^(13a)), and C₁₋₆        alkyl; wherein C₁₋₆ alkyl is optionally substituted with one or        more halogen, which are the same or different;    -   each —R¹², —R^(12a), —R¹³, —R^(13a), —R^(13b) is independently        selected from the group consisting of —H, and C₁₋₆ alkyl;        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different;    -   optionally, one or more of the pairs —R¹/—R^(1a), —R²/—R^(2a),        —R³/—R^(3a), —R⁶/—R^(6a), —R⁷/—R^(7a) are joined together with        the atom to which they are attached to form a C₃₋₁₀ cycloalkyl        or a 3- to 10-membered heterocyclyl;    -   optionally, one or more of the pairs —R¹/—R², —R¹/—R³, —R¹/—R⁴,        —R¹/—R⁵, —R¹/—R⁶, —R¹/—R⁷, —R²/—R³, —R²/—R⁴, —R²/—R⁵, —R²/—R⁶,        —R²/—R⁷, —R³/—R⁴, —R³/—R⁵, —R³/—R⁶, —R³/—R⁷, —R⁴/—R⁵, —R⁴/—R⁶,        —R⁴/—R⁷, —R⁵/—R⁶, —R⁵/—R⁷, —R⁶/—R⁷ are joint together with the        atoms to which they are attached to form a ring A;    -   A is selected from the group consisting of phenyl; naphthyl;        indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 3- to        10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;    -   wherein    -   -L²- is a single chemical bond or a spacer;    -   —Z is a water-soluble carrier; and    -   -Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (III) arepreferably as described above.

Preferably -L¹- of formula (III) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (III) is not further substituted.

Additional preferred embodiments for -L¹- are disclosed in EP1536334B1,WO2009/009712A1, WO2008/034122A1, WO2009/143412A2, WO2011/082368A2, andU.S. Pat. No. 8,618,124B2, which are herewith incorporated by referencein their entirety.

Additional preferred embodiments for -L¹- are disclosed in U.S. Pat. No.8,946,405B2 and U.S. Pat. No. 8,754,190B2, which are herewithincorporated by reference in their entirety. Accordingly, a preferredmoiety -L¹- is of formula (IV):

-   -   wherein    -   the dashed line indicates attachment to -D which is a PTH moiety        and wherein attachment is through a functional group of -D        selected from the group consisting of —OH, —SH and —NH₂;    -   m is 0 or 1;    -   at least one or both of —R¹ and —R² is/are independently of each        other selected from the group consisting of —CN, —NO₂,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted alkenyl, optionally substituted alkynyl,        —C(O)R³, —S(O)R³, —S(O)₂R³, and —SR⁴,    -   one and only one of —R¹ and —R² is selected from the group        consisting of —H, optionally substituted alkyl, optionally        substituted arylalkyl, and optionally substituted        heteroarylalkyl;    -   —R³ is selected from the group consisting of —H, optionally        substituted alkyl, optionally substituted aryl, optionally        substituted arylalkyl, optionally substituted heteroaryl,        optionally substituted heteroarylalkyl, —OR⁹ and —N(R⁹)₂;    -   —R⁴ is selected from the group consisting of optionally        substituted alkyl, optionally substituted aryl, optionally        substituted arylalkyl, optionally substituted heteroaryl, and        optionally substituted heteroarylalkyl;    -   each —R⁵ is independently selected from the group consisting of        —H, optionally substituted alkyl, optionally substituted        alkenylalkyl, optionally substituted alkynylalkyl, optionally        substituted aryl, optionally substituted arylalkyl, optionally        substituted heteroaryl and optionally substituted        heteroarylalkyl;    -   —R⁹ is selected from the group consisting of —H and optionally        substituted alkyl;    -   —Y— is absent and —X— is —O— or —S—; or    -   —Y— is —N(Q)CH₂— and —X— is —O—;    -   Q is selected from the group consisting of optionally        substituted alkyl, optionally substituted aryl, optionally        substituted arylalkyl, optionally substituted heteroaryl and        optionally substituted heteroarylalkyl;    -   optionally, —R and —R² may be joined to form a 3 to 8-membered        ring; and    -   optionally, both —R⁹ together with the nitrogen to which they        are attached form a heterocyclic ring;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;    -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   -Z′ is a water-insoluble carrier.

Only in the context of formula (IV) the terms used have the followingmeaning: The term “alkyl” as used herein includes linear, branched orcyclic saturated hydrocarbon groups of 1 to 8 carbons, or in someembodiments 1 to 6 or 1 to 4 carbon atoms.

The term “alkoxy” includes alkyl groups bonded to oxygen, includingmethoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.

The term “alkenyl” includes non-aromatic unsaturated hydrocarbons withcarbon-carbon double bonds.

The term “alkynyl” includes non-aromatic unsaturated hydrocarbons withcarbon-carbon triple bonds.

The term “aryl” includes aromatic hydrocarbon groups of 6 to 18 carbons,preferably 6 to 10 carbons, including groups such as phenyl, naphthyl,and anthracenyl. The term “heteroaryl” includes aromatic ringscomprising 3 to 15 carbons containing at least one N, O or S atom,preferably 3 to 7 carbons containing at least one N, O or S atom,including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl,indenyl, and similar.

In some instance, alkenyl, alkynyl, aryl or heteroaryl moieties may becoupled to the remainder of the molecule through an alkylene linkage.Under those circumstances, the substituent will be referred to asalkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicatingthat an alkylene moiety is between the alkenyl, alkynyl, aryl orheteroaryl moiety and the molecule to which the alkenyl, alkynyl, arylor heteroaryl is coupled.

The term “halogen” includes bromo, fluoro, chloro and iodo.

The term “heterocyclic ring” refers to a 4 to 8 membered aromatic ornon-aromatic ring comprising 3 to 7 carbon atoms and at least one N, O,or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl,pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groupsprovided for the term “heteroaryl” above.

When a ring system is optionally substituted, suitable substituents areselected from the group consisting of alkyl, alkenyl, alkynyl, or anadditional ring, each optionally further substituted. Optionalsubstituents on any group, including the above, include halo, nitro,cyano, —OR, —SR, —NR₂, —OCOR, —NRCOR, —COOR, —CONR₂, —SOR, —SO₂R,—SONR₂, —SO₂N R₂, wherein each R is independently alkyl, alkenyl,alkynyl, aryl or heteroaryl, or two R groups taken together with theatoms to which they are attached form a ring.

Preferably -L¹- of formula (IV) is substituted with one moiety -L²-Z or-L²-Z′.

An additional preferred embodiment for -L¹- is disclosed inWO2013/036857A1, which is herewith incorporated by reference in itsentirety. Accordingly, a preferred moiety -L¹- is of formula (V):

-   -   wherein    -   the dashed line indicates attachment to -D which is a PTH moiety        and wherein attachment is through an amine functional group of        -D;    -   —R¹ is selected from the group consisting of optionally        substituted C₁-C₆ linear, branched, or cyclic alkyl; optionally        substituted aryl; optionally substituted heteroaryl; alkoxy; and        —NR⁵²;    -   —R² is selected from the group consisting of —H; optionally        substituted C₁-C₆ alkyl; optionally substituted aryl; and        optionally substituted heteroaryl;    -   —R³ is selected from the group consisting of —H; optionally        substituted C₁-C₆ alkyl; optionally substituted aryl; and        optionally substituted heteroaryl;    -   —R⁴ is selected from the group consisting of —H; optionally        substituted C₁-C₆ alkyl; optionally substituted aryl; and        optionally substituted heteroaryl;    -   each —R⁵ is independently of each other selected from the group        consisting of —H; optionally substituted C₁-C₆ alkyl; optionally        substituted aryl; and optionally substituted heteroaryl; or when        taken together two —R⁵ can be cycloalkyl or cycloheteroalkyl;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;    -   wherein    -   -L²- is a single chemical bond or a spacer;    -   —Z is a water-soluble carrier; and    -   -Z′ is a water-insoluble carrier.

Only in the context of formula (V) the terms used have the followingmeaning:

“Alkyl”, “alkenyl”, and “alkynyl” include linear, branched or cyclichydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4 carbons whereinalkyl is a saturated hydrocarbon, alkenyl includes one or morecarbon-carbon double bonds and alkynyl includes one or morecarbon-carbon triple bonds. Unless otherwise specified these contain 1-6C.

“Aryl” includes aromatic hydrocarbon groups of 6-18 carbons, preferably6-10 carbons, including groups such as phenyl, naphthyl, and anthracene“Heteroaryl” includes aromatic rings comprising 3-15 carbons containingat least one N, O or S atom, preferably 3-7 carbons containing at leastone N, O or S atom, including groups such as pyrrolyl, pyridyl,pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiszolyl, isothiazolyl,quinolyl, indolyl, indenyl, and similar.

The term “substituted” means an alkyl, alkenyl, alkynyl, aryl, orheteroaryl group comprising one or more substituent groups in place ofone or more hydrogen atoms. Substituents may generally be selected fromhalogen including F, Cl, Br, and I; lower alkyl including linear,branched, and cyclic; lower haloalkyl including fluoroalkyl,chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy includinglinear, branched, and cyclic; SH; lower alkylthio including linear,branched and cyclic; amino, alkylamino, dialkylamino, silyl includingalkylsilyl, alkoxysilyl, and arylsilyl; nitro; cyano; carbonyl;carboxylic acid, carboxylic ester, carboxylic amide, aminocarbonyl;aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketne; sulfone;sulfonamide; aryl including phenyl, naphthyl, and anthracenyl;heteroaryl including 5-member heteroaryls including as pyrrole,imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole,thiadiazole, triazole, oxadiazole, and tetrazole, 6-member heteroarylsincluding pyridine, pyrimidine, pyrazine, and fused heteroarylsincluding benzofuran, benzothiophene, benzoxazole, benzimidazole,indole, benzothiazole, benzisoxazole, and benzisothiazole.

Preferably -L¹- of formula (V) is substituted with one moiety -L²-Z or-L²-Z′.

A further preferred embodiment for -L¹- is disclosed in U.S. Pat. No.7,585,837B2, which is herewith incorporated by reference in itsentirety. Accordingly, a preferred moiety -L¹- is of formula (VI):

-   -   wherein    -   the dashed line indicates attachment to -D which is a PTH moiety        and wherein attachment is through an amine functional group of        -D;    -   R¹ and R² are independently selected from the group consisting        of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl,        halogen, nitro, —SO₃H, —SO₂NHR⁵, amino, ammonium, carboxyl,        PO₃H₂, and OPO₃H₂;    -   R³, R⁴, and R⁵ are independently selected from the group        consisting of hydrogen, alkyl, and aryl;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   -Z′ is a water-insoluble carrier.

Suitable substituents for formulas (VI) are alkyl (such as C₁₋₆ alkyl),alkenyl (such as C₂₋₆ alkenyl), alkynyl (such as C₂₋₆ alkynyl), aryl(such as phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl(such as aromatic 4 to 7 membered heterocycle) or halogen moieties.

Only in the context of formula (VI) the terms used have the followingmeaning:

The terms “alkyl”, “alkoxy”, “alkoxyalkyl”, “aryl”, “alkaryl” and“aralkyl” mean alkyl radicals of 1-8, preferably 1-4 carbon atoms, e.g.methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals of 6-10carbon atoms, e.g. phenyl and naphthyl. The term “halogen” includesbromo, fluoro, chloro and iodo.

Preferably -L¹- of formula (VI) is substituted with one moiety -L²-Z or-L²-Z′.

A further preferred embodiment for -L¹- is disclosed in WO2002/089789A1,which is herewith incorporated by reference in its entirety.Accordingly, a preferred moiety -L¹- is of formula (VII):

-   -   wherein    -   the dashed line indicates attachment to -D which is a PTH moiety        and wherein attachment is through an amine functional group of        -D;    -   L₁ is a bifunctional linking group,    -   Y₁ and Y₂ are independently 0, S or NR¹;    -   R², R³, R⁴, R⁵, R⁶ and R⁷ are independently selected from the        group consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched        alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈        substituted cycloalkyls, aryls, substituted aryls, aralkyls,        C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy,        phenoxy, and C₁₋₆ heteroalkoxy;    -   Ar is a moiety which when included in formula (VII) forms a        multisubstituted aromatic hydrocarbon or a multi-substituted        heterocyclic group;    -   X is a chemical bond or a moiety that is actively transported        into a target cell, a hydrophobic moiety, or a combination        thereof,    -   y is 0 or 1;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   -Z′ is a water-insoluble carrier.

Only in the context of formula (VII) the terms used have the followingmeaning:

The term “alkyl” shall be understood to include, e.g. straight,branched, substituted C₁₋₁₂ alkyls, including alkoxy, C₃₋₈ cycloalkylsor substituted cycloalkyls, etc. The term “substituted” shall beunderstood to include adding or replacing one or more atoms containedwithin a functional group or compounds with one or more different atoms.

Substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos,hydroxyalkyls and mercaptoalkyls; substituted cycloalkyls includemoieties such as 4-chlorocyclohexyl; aryls include moieties such asnapthyl; substituted aryls include moieties such as 3-bromo-phenyl;aralkyls include moieties such as toluyl; heteroalkyls include moietiessuch as ethylthiophene; substituted heteroalkyls include moieties suchas 3-methoxythiophone; alkoxy includes moieties such as methoxy; andphenoxy includes moieties such as 3-nitrophenoxy. Halo-shall beunderstood to include fluoro, chloro, iodo and bromo.

Preferably -L¹- of formula (VII) is substituted with one moiety -L²-Z or-L²-Z′.

In another preferred embodiment -L¹- comprises a substructure of formula(VIII)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        a nitrogen of -D which is a PTH moiety by forming an amide bond;    -   the unmarked dashed lines indicate attachment to the remainder        of -L¹-; and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   -Z′ is a water-insoluble carrier.

Preferably -L¹- of formula (VIII) is substituted with one moiety -L²-Zor -L²-Z′.

In one embodiment -L¹- of formula (VIII) is not further substituted.

In another preferred embodiment -L¹- comprises a substructure of formula(IX)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        a nitrogen of -D which is a PTH moiety by forming a carbamate        bond;    -   the unmarked dashed lines indicate attachment to the remainder        of -L¹-; and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   -Z′ is a water-insoluble carrier.

Preferably -L¹- of formula (IX) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (IX) is not further substituted.

In the prodrugs of the present invention -L²- is a chemical bond or aspacer moiety.

In one embodiment -L²- is a chemical bond.

In another embodiment -L²- is a spacer moiety.

When -L²- is other than a single chemical bond, -L²- is preferablyselected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(y1))—, —S(O)₂N(R^(y1))—, —S(O)N(R^(y1))—, —S(O)₂—, —S(O)—,—N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—,—N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl,and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally substituted with one or more —R², which are thesame or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—,—S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R^(y3))—;

—R^(y1) and —R^(y1a) are independently of each other selected from thegroup consisting of —H, -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl; wherein -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl areoptionally substituted with one or more —R^(y2), which are the same ordifferent, and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl areoptionally interrupted by one or more groups selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y4))—,—S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—, —S(O)₂—, —S(O)—,—N(R^(y4))S(O)₂N(R^(y4a)), —S—, —N(R^(y4))—, —OC(OR⁴)(R^(y4a))—,—N(R⁴)C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R², which are the same or different;

each —R^(y2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(y5), —OR^(y5), —C(O)R^(y5),—C(O)N(R^(y5)R^(y5a)), —S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)),—S(O)₂R^(y5), —S(O)R^(y5), —N(R^(Y5))S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(Y5)R^(y5a)), —NO₂, —OC(O)R^(y5), —N(R^(y5))C(O)R^(y5a),—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5a),—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and

each —R^(y3), —R^(y3a), —R⁴, —R^(y4a), —R⁵, —R^(y5a) and —R^(y5b) isindependently selected from the group consisting of —H, and C₁₋₆ alkyl,wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different.

When -L²- is other than a single chemical bond, -L²- is even morepreferably selected from -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y1))—,—S(O)₂N(R^(y1))—, —S(O)N(R^(y1))—, —S(O)₂—, —S(O)—,—N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—,—N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl,and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀alkynyl are optionally substituted with one or more —R², which are thesame or different and wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—,—S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R^(y3))—;

—R^(y1) and —R^(y1a) are independently of each other selected from thegroup consisting of —H, -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀alkynyl; wherein -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl areoptionally substituted with one or more —R², which are the same ordifferent, and wherein C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl areoptionally interrupted by one or more groups selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y4))—,—S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—, —S(O)₂—, —S(O)—,—N(R^(y4))S(O)₂N(R^(y4a)), —S—, —N(R^(y4))—, —OC(OR^(y4))(R^(y4a))—,—N(R^(y4))C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R², which are the same or different;

—R^(y2) is selected from the group consisting of halogen, —CN, oxo (═O),—COOR^(y5), —OR^(y5), —C(O)R^(y5), —C(O)N(R^(y5)R^(y5a)),—S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)), —S(O)₂R^(y5),—S(O)R^(y5), —N(R^(y5))S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(y5)R^(y5a)), —NO₂, —OC(O)R^(y5), —N(R^(y5)) C(O)R^(y5a),—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5a),—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(Y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R⁵, —R^(y5a) and —R^(y5b) isindependently of each other selected from the group consisting of —H,and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different.

When -L²- is other than a single chemical bond, -L²- is even morepreferably selected from the group consisting of -T-, —C(O)O—, —O—,—C(O)—, —C(O)N(R^(y1))—, —S(O)₂N(R^(y1))—, —S(O)N(R^(y1))—, —S(O)₂—,—S(O)—, —N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—,—OC(OR^(y1))(R^(y1a))—, —N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—,C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₅₀ alkyl,C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally substituted with one ormore —R², which are the same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl are optionally interrupted by one or moregroups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(y3))—, —S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R^(y3))—;

—R^(y1) and —R^(y1a) are independently selected from the groupconsisting of —H, -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;

each —R^(y2) is independently selected from the group consisting ofhalogen, and C₁₋₆ alkyl; and each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a),—R^(y5), —R^(y5a) and —R^(y5b) is independently of each other selectedfrom the group consisting of —H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent.

Even more preferably, -L²- is a C₁₋₂₀ alkyl chain, which is optionallyinterrupted by one or more groups independently selected from —O—, -T-and —C(O)N(R^(y1))—; and which C₁₋₂₀ alkyl chain is optionallysubstituted with one or more groups independently selected from —OH, -Tand —C(O)N(R^(y6)R^(y6a)); wherein —R¹, —R^(y6), —R^(y6a) areindependently selected from the group consisting of H and C₁₋₄ alkyl andwherein T is selected from the group consisting of phenyl, naphthyl,indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to 10-memberedheterocyclyl, 8- to 11-membered heterobicyclyl, 8-to 30-memberedcarbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

Preferably, -L²- has a molecular weight in the range of from 14 g/mol to750 g/mol.

Preferably, -L²-comprises a moiety selected from

wherein

dashed lines indicate attachment to the rest of -L²-, -L¹-, —Z and/or-Z′, respectively; and

—R and —R^(a) are independently of each other selected from the groupconsisting of —H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

In one preferred embodiment -L²- has a chain lengths of 1 to 20 atoms.

As used herein the term “chain length” with regard to the moiety -L²-refers to the number of atoms of -L²- present in the shortest connectionbetween -L¹- and -Z.

Preferably, -L²- is of formula (i)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        -L¹-;    -   the unmarked dashed line indicates attachment to -Z or -Z′;    -   n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18; and    -   wherein the moiety of formula (i) is optionally further        substituted.

Preferably, n of formula (i) is selected from the group consisting of 3,4, 5, 6, 7, 8, and 9. Even more preferably n of formula (i) is 4, 5, 6,or 7. In one embodiment n of formula (i) is 4. In another embodiment nof formula (i) is 5. In another embodiment n of formula (i) is 6.

In one preferred embodiment the moiety -L-L²- is selected from the groupconsisting of

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        -Z or -Z′.

In one preferred embodiment the moiety -L¹-L²- is selected from thegroup consisting of

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        -Z or -Z′.

In a preferred embodiment the moiety -L¹-L²- is of formula (IIca-ii).

In another preferred embodiment the moiety -L¹-L²- is of formula(IIcb-iii).

Preferably, the PTH prodrug of the present invention is of formula (Ia)with x=1.

The carrier -Z comprises a C₈₋₂₄ alkyl or a polymer. Preferably, —Zcomprises a polymer, preferably a polymer selected from the groupconsisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylicacids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

Preferably, —Z has a molecular weight ranging from 5 to 200 kDa. Evenmore preferably, —Z has a molecular weight ranging from 8 to 100 kDa,even more preferably ranging from 10 to 80 kDa, even more preferablyfrom 12 to 60, even more preferably from 15 to 40 and most preferably —Zhas a molecular weight of about 20 kDa. In another equally preferredembodiment -Z has a molecular weight of about 40 kDa.

In one embodiment such water-soluble carrier -Z comprises a protein.Preferred proteins are selected from the group consisting ofcarboxyl-terminal polypeptide of the chorionic gonadotropin as describedin US 2012/0035101 A1 which are herewith incorporated by reference;albumin; XTEN sequences as described in WO 2011123813 A2 which areherewith incorporated by reference; proline/alanine random coilsequences as described in WO 2011/144756 A1 which are herewithincorporated by reference; proline/alanine/serine random coil sequencesas described in WO 2008/155134 A1 and WO 2013/024049 A1 which areherewith incorporated by reference; and Fc fusion proteins.

In one embodiment -Z is a polysarcosine.

In another preferred embodiment -Z comprises a poly(N-methylglycine).

In a particularly preferred embodiment -Z comprises a random coilprotein moiety.

In one preferred embodiment -Z comprises one random coil protein moiety.

In another preferred embodiment -Z comprises two random coil proteinsmoieties.

In another preferred embodiment -Z comprises three random coil proteinsmoieties.

In another preferred embodiment -Z comprises four random coil proteinsmoieties.

In another preferred embodiment -Z comprises five random coil proteinsmoieties.

In another preferred embodiment -Z comprises six random coil proteinsmoieties.

In another preferred embodiment -Z comprises seven random coil proteinsmoieties.

In another preferred embodiment -Z comprises eight random coil proteinsmoieties.

Preferably such random coil protein moiety comprises at least 25 aminoacid residues and at most 2000 amino acids. Even more preferably suchrandom coil protein moiety comprises at least 30 amino acid residues andat most 1500 amino acid residues. Even more preferably such random coilprotein moiety comprises at least 50 amino acid residues and at most 500amino acid residues.

In a preferred embodiment, —Z comprises a random coil protein moiety ofwhich at least 80%, preferably at least 85%, even more preferably atleast 90%, even more preferably at least 95%, even more preferably atleast 98% and most preferably at least 99% of the total number of aminoacids forming said random coil protein moiety are selected from alanineand proline. Even more preferably, at least 10%, but less than 75%,preferably less than 65%, of the total number of amino acid residues ofsuch random coil protein moiety are proline residues. Preferably, suchrandom coil protein moiety is as described in WO 2011/144756 A1 which ishereby incorporated by reference in its entirety. Even more preferably—Z comprises at least one moiety selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:51 and SEQ ID NO:61 as disclosed inWO2011/144756 which are hereby incorporated by reference. A moietycomprising such random coil protein comprising alanine and proline willbe referred to as “PA” or “PA moiety”.

Accordingly, —Z comprises a PA moiety.

In an equally preferred embodiment, —Z comprises a random coil proteinmoiety of which at least 80%, preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95%, even morepreferably at least 98% and most preferably at least 99% of the totalnumber of amino acids forming said random coil protein moiety areselected from alanine, serine and proline. Even more preferably, atleast 4%, but less than 40% of the total number of amino acid residuesof such random coil protein moiety are proline residues. Preferably,such random coil protein moiety is as described in WO 2008/155134 A1which is hereby incorporated by reference in its entirety. Even morepreferably —Z comprises at least one moiety selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54 and SEQ ID NO:56 as disclosed in WO 2008/155134 A1, which arehereby incorporated by reference. A moiety comprising such random coilprotein moiety comprising alanine, serine and proline will be referredto as “PAS” or “PAS moiety”.

Accordingly, —Z comprises a PAS moiety.

In an equally preferred embodiment, —Z comprises a random coil proteinmoiety of which at least 80%, preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95%, even morepreferably at least 98% and most preferably at least 99% of the totalnumber of amino acids forming said random coil protein moiety areselected from alanine, glycine and proline. A moiety comprising suchrandom coil protein moiety comprising alanine, glycine and proline willbe referred to as “PAG” or “PAG moiety”.

Accordingly, —Z comprises a PAG moiety.

In an equally preferred embodiment, —Z comprises a random coil proteinmoiety of which at least 80%, preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95%, even morepreferably at least 98% and most preferably at least 99% of the totalnumber of amino acids forming said random coil protein moiety areselected from proline and glycine. A moiety comprising such random coilprotein moiety comprising proline and glycine will be referred to as“PG” or “PG moiety”.

Preferably, such PG moiety comprises a moiety of formula (a-0)

-   -   [(Gly)_(p)-Pro-(Gly)_(q)]_(r) (a-0);    -   wherein    -   p is selected from the group consisting of 0, 1, 2, 3, 4 and 5;    -   q is selected from the group consisting of 0, 1, 2, 3, 4 and 5;    -   r is an integer ranging from and including 10 to 1000;    -   provided that at least one of p and q is at least 1;

Preferably, p of formula (a-0) is selected from the group consisting of1, 2 and 3.

Preferably, q of formula (a-0) is selected from 0, 1 and 2.

Even more preferably the PG moiety comprises the sequence of SEQ ID:NO122: GGPGGPGPGGPGGPGPGGPG

Even more preferably, the PG moiety comprises the sequence of SEQ ID:NO97 of formula (a-0-a)

-   -   (GGPGGPGPGGPGGPGPGGPG)_(v) (a-0-a),    -   wherein    -   v is an integer ranging from and including 1 to 50.

Accordingly, —Z comprises a PG moiety.

In an equally preferred embodiment, —Z comprises a random coil proteinmoiety of which at least 80%, preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95%, even morepreferably at least 98% and most preferably at least 99% of the totalnumber of amino acids forming said random coil protein moiety areselected from alanine, glycine, serine, threonine, glutamate andproline. Preferably, such random coil protein moiety is as described inWO 2010/091122 A1 which is hereby incorporated by reference. Even morepreferably —Z comprises at least one moiety selected from the groupconsisting of SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184; SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ IDNO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ IDNO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:759,SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ IDNO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQID NO:769, SEQ ID NO:770, SEQ ID NO:771, SEQ ID NO:772, SEQ ID NO:773,SEQ ID NO:774, SEQ ID NO:775, SEQ ID NO:776, SEQ ID NO:777, SEQ IDNO:778, SEQ ID NO:779, SEQ ID NO:1715, SEQ ID NO:1716, SEQ ID NO:1718,SEQ ID NO:1719, SEQ ID NO:1720, SEQ ID NO:1721 and SEQ ID NO:1722 asdisclosed in WO2010/091122A1, which are hereby incorporated byreference. A moiety comprising such random coil protein moietycomprising alanine, glycine, serine, threonine, glutamate and prolinewill be referred to as “XTEN” or “XTEN moiety” in line with itsdesignation in WO 2010/091122 A1.

Accordingly, —Z comprises an XTEN moiety.

In another preferred embodiment, —Z comprises a fatty acid derivate.Preferred fatty acid derivatives are those disclosed in WO 2005/027978A2 and WO 2014/060512 A1 which are herewith incorporated by reference.

In another preferred embodiment -Z is a hyaluronic acid-based polymer.

In one embodiment -Z is a carrier as disclosed in WO 2012/02047 A1 whichis herewith incorporated by reference.

In another embodiment -Z is a carrier as disclosed in WO 2013/024048 A1which is herewith incorporated by reference.

In another preferred embodiment -Z is a PEG-based polymer, such as alinear, branched or multi-arm PEG-based polymer.

In one embodiment -Z is a linear PEG-based polymer.

In another embodiment -Z is a multi-arm PEG-based polymer. Preferably,—Z is a multi-arm PEG-based polymer having at least 4 PEG-based arms.

Preferably, such multi-arm PEG-based polymer -Z is connected to amultitude of moieties -L²-L¹-D, wherein each moiety -L²-L¹-D ispreferably connected to the end of an arm, preferably to the end of anarm. Preferably such multi-arm PEG-based polymer -Z is connected to 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 moieties -L²-L¹-D.Even more preferably such multi-arm PEG-based polymer -Z is connected to2, 3, 4, 6 or 8 moieties -L²-L¹-D. Even more preferably such multi-armPEG-based polymer -Z is connected to 2, 4 or 6 moieties -L²-L-D, evenmore preferably such multi-arm PEG-based polymer -Z is connected to 4 or6 moieties -L²-L¹-D, and most preferably such multi-arm PEG-basedpolymer -Z is connected to 4 moieties -L²-L¹-D.

Preferably, such multi-arm PEG-based polymer -Z is a multi-arm PEGderivative as, for instance, detailed in the products list of JenKemTechnology, USA (accessed by download fromhttp://www.jenkemusa.com/Pages/PEGProducts.aspx on Dec. 18, 2014), suchas a 4-arm-PEG derivative, in particular a 4-arm-PEG comprising apentaerythritol core, an 8-arm-PEG derivative comprising a hexaglycerincore, and an 8-arm-PEG derivative comprising a tripentaerythritol core.More preferably, the water-soluble PEG-based carrier -Z comprises amoiety selected from:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 20 to 500;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin or tripentaerythritol core structure; and

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=comprising a sorbitol or dipentaerythritol core;

and wherein dashed lines indicate attachment to the rest of the PTHprodrug.

In a preferred embodiment -Z is a branched PEG-based polymer. In oneembodiment -Z is a branched PEG-based polymer having one, two, three,four, five or six branching points. Preferably, —Z is a branchedPEG-based polymer having one, two or three branching points. In oneembodiment -Z is a branched PEG-based polymer having one branchingpoint. In another embodiment -Z is a branched PEG-based polymer havingtwo branching points. In another embodiment -Z is a branched PEG-basedpolymer having three branching points. A branching point is preferablyselected from the group consisting of —N<, —CH< and >C<.

Preferably, such branched PEG-based moiety —Z has a molecular weight ofat least 10 kDa.

In one embodiment such branched moiety —Z has a molecular weight rangingfrom and including 10 kDa to 500 kDa, more preferably ranging from andincluding 10 kDa to 250 Da, even more preferably ranging from andincluding 10 kDa to 150 kDa, even more preferably ranging from andincluding 12 kDa to 100 kDa and most preferably ranging from andincluding 15 kDa to 80 kDa.

Preferably, such branched moiety —Z has a molecular weight ranging fromand including 10 kDa to 80 kDa. In one embodiment the molecular weightis about 10 kDa. In another embodiment the molecular weight of suchbranched moiety —Z is about 20 kDa. In another embodiment the molecularweight of such branched moiety —Z is about 30 kDa. In another embodimentthe molecular weight of such a branched moiety —Z is about 40 kDa. Inanother embodiment the molecular weight of such a branched moiety —Z isabout 50 kDa. In another embodiment the molecular weight of such abranched moiety —Z is about 60 kDa. In another embodiment the molecularweight of such a branched moiety —Z is about 70 kDa. In anotherembodiment the molecular weight of such a branched moiety —Z is about 80kDa. Most preferably, such branched moiety —Z has a molecular weight ofabout 40 kDa.

Preferably, —Z or -Z′ comprises a moiety

In an equally preferred embodiment -Z comprises an amide bond.

Preferably -Z comprises a moiety of formula (a)

-   -   wherein    -   the dashed line indicates attachment to -L²- or to the remainder        of -Z;    -   BP^(a) is a branching point selected from the group consisting        of —N<, —CR< and >C<;    -   —R is selected from the group consisting of —H and C₁₋₆ alkyl;    -   a is 0 if BP^(a) is —N< or —CR< and n is 1 if BP^(a) is >C<;    -   —S^(a)—, S^(a′)—, —S^(a″)— and —S^(a′″)— are independently of        each other a chemical bond or are selected from the group        consisting of C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl;        wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are        optionally substituted with one or more —R¹, which are the same        or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R²)—, —S(O)₂N(R²)—, —S(O)N(R²)—, —S(O)₂—, —S(O)—,        —N(R²)S(O)₂N(R^(2a))—, —S—, —N(R²)—, —OC(OR₂)(R^(2a))—,        —N(R²)C(O)N(R^(2a))—, and —OC(O)N(R²)—;    -   each -T- is independently selected from the group consisting of        phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀        cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered        heterobicyclyl, 8-to 30-membered carbopolycyclyl, and 8- to        30-membered heteropolycyclyl; wherein each -T- is independently        optionally substituted with one or more —R¹, which are the same        or different;    -   each —R¹ is independently selected from the group consisting of        halogen, —CN, oxo (═O), —COOR³, —OR³, —C(O)R³, —C(O)N(R³R^(3a)),        —S(O)₂N(R³R^(3a)), —S(O)N(R³R^(3a)), —S(O)₂R³, —S(O)R³,        —N(R³)S(O)₂N(R^(3a)R^(3b)), —SR³, —N(R³R^(3a)), —NO₂, —OC(O)R³,        —N(R³)C(O)R^(3a), —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a),        —N(R³)C(O)OR^(3a), —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)),        and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionally substituted        with one or more halogen, which are the same or different; each        —R², —R^(2a), —R³, —R^(3a) and —R^(3b) is independently selected        from the group consisting of —H, and C₁₋₆ alkyl, wherein C₁₋₆        alkyl is optionally substituted with one or more halogen, which        are the same or different; and    -   -P^(a′), -P^(a″) and -P^(a′″) are independently a polymeric        moiety.

In one embodiment BP^(a) of formula (a) is —N<.

In another embodiment BP^(a) of formula (a) is >C<.

In a preferred embodiment BP^(a) of formula (a) is —CR<. Preferably, —Ris —H. Accordingly, a of formula (a) is 0.

In one embodiment —S^(a)— of formula (a) is a chemical bond.

In another embodiment —S^(a)— of formula (a) is selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, which C₁₋₁₀alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interrupted by oneor more chemical groups selected from the group consisting of -T-,—C(O)O—, —O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—,—S(O)—, —N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein -T- is a 3- to10-membered heterocyclyl; and —R⁴ and —R^(4a) are independently selectedfrom the group consisting of —H, methyl, ethyl, propyl and butyl.

Preferably —S^(a)— of formula (a) is selected from the group consistingof C₁₋₁₀ alkyl which is interrupted by one or more chemical groupsselected from the group consisting of -T-, —C(O)N(R⁴)— and —O—.

In one embodiment —S^(a′)— of formula (a) is a chemical bond.

In another embodiment —S^(a)— of formula (a) is selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, which C₁₋₁₀alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interrupted by oneor more chemical groups selected from the group consisting of —C(O)O—,—O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—, —S(O)—,—N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a′)— of formula (a) is selected fromthe group consisting of methyl, ethyl, propyl, butyl, which areoptionally interrupted by one or more chemical groups selected from thegroup consisting of —O—, —C(O)— and —C(O)N(R⁴)—.

In one embodiment —S^(a″)— of formula (a) is a chemical bond.

In another embodiment —S^(a″)— of formula (a) is selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, which C₁₋₁₀alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interrupted by oneor more chemical groups selected from the group consisting of —C(O)O—,—O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—, —S(O)—,—N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a″)— of formula (a) is selected fromthe group consisting of methyl, ethyl, propyl, butyl, which areoptionally interrupted by one or more chemical groups selected from thegroup consisting of —O—, —C(O)— and —C(O)N(R⁴)—.

In one embodiment —S^(a′″)— of formula (a) is a chemical bond.

In another embodiment —S^(a′″)— of formula (a) is selected from thegroup consisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, whichC₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interruptedby one or more chemical groups selected from the group consisting of—C(O)O—, —O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—,—S(O)—, —N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a′″)— of formula (a) is selected fromthe group consisting of methyl, ethyl, propyl, butyl, which areoptionally interrupted by one or more chemical groups selected from thegroup consisting of —O—, —C(O)— and —C(O)N(R⁴)—.

Preferably, -P^(a′)-P^(a″) and -P^(a′″) of formula (a) independentlycomprise a polymer selected from the group consisting of2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

More preferably, -P^(a′)-P^(a″) and -P^(a′″) of formula (a)independently comprise a PEG-based moiety. Even more preferably,-P^(a′)-P^(a″) and -P^(a′″) of formula (a) independently comprise aPEG-based moiety comprising at least 20% PEG, even more preferably atleast 30%, even more preferably at least 40% PEG, even more preferablyat least 50% PEG, even more preferably at least 60% PEG, even morepreferably at least 70% PEG, even more preferably at least 80% PEG andmost preferably at least 90% PEG.

Preferably, -P^(a′)-P^(a″) and -P^(a′″) of formula (a) independentlyhave a molecular weight ranging from and including 5 kDa to 50 kDa, morepreferably have a molecular weight ranging from and including 5 kDa to40 kDa, even more preferably ranging from and including 7.5 kDa to 35kDa, even more preferably ranging from and 7.5 to 30 kDa, even morepreferably ranging from and including 10 to 30 kDa.

In one embodiment -P^(a′)-P^(a″) and -P^(a′″) of formula (a) have amolecular weight of about 5 kDa.

In another embodiment -P^(a′)-P^(a″) and -P^(a′″) of formula (a) have amolecular weight of about 7.5 kDa.

In another embodiment -P^(a′)-P^(a″) and -P^(a′″) of formula (a) have amolecular weight of about 10 kDa.

In another embodiment -P^(a′)-P^(a″) and -P^(a′″) of formula (a) have amolecular weight of about 12.5 kDa.

In another embodiment -P^(a′)-P^(a″) and -P^(a′″) of formula (a) have amolecular weight of about 15 kDa.

In another embodiment -P^(a′)-P^(a″) and -P^(a′″) of formula (a) have amolecular weight of about 20 kDa.

In one embodiment -Z comprises one moiety of formula (a).

In another embodiment -Z comprises two moieties of formula (a).

In another embodiment -Z comprises three moieties of formula (a).

Preferably, —Z is a moiety of formula (a).

More preferably, —Z comprises a moiety of formula (b)

-   -   wherein    -   the dashed line indicates attachment to -L²- or to the remainder        of -Z; and    -   m and p are independently of each other an integer ranging from        and including 150 to 1000; preferably an integer ranging from        and including 150 to 500; more preferably an integer ranging        from and including 200 to 500; and most preferably an integer        ranging from and including 400 to 500.

Preferably, m and p of formula (b) are the same integer.

Most preferably m and p of formula (b) are about 450.

Preferably, —Z is a moiety of formula (b).

The carrier -Z′ is a water-insoluble polymer, even more preferably ahydrogel. Preferably, such hydrogel comprises a polymer selected fromthe group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins,poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy)polymers, poly(amides), poly(amidoamines), poly(amino acids),poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolicacids), polybutylene terephthalates, poly(caprolactones),poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides),poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethyleneoxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolicacids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

If the carrier -Z′ is a hydrogel, it is preferably a hydrogel comprisingPEG or hyaluronic acid. Most preferably such hydrogel comprises PEG.

Even more preferably, the carrier -Z′ is a hydrogel as described in WO2006/003014 A2, WO 2011/012715 A1 or WO 2014/056926 A1, which areherewith incorporated by reference in their entirety.

In another embodiment -Z′ is a polymer network formed through thephysical aggregation of polymer chains, which physical aggregation ispreferably caused by hydrogen bonds, crystallization, helix formation orcomplexation. In one embodiment such polymer network is a thermogellingpolymer.

Preferably, the total mass of the PTH prodrug of the present inventionis at least 10 kDa, such as at least 12 kDa, such as at least 15 kDa,such as at least 20 kDa or such as at least 30 kDa. It is preferred thatthe total mass of the PTH prodrug of the present invention is at most250 kDa, such as at most 200 kDa, 180 kDa, 150 kDa or 100 kDa.

In a preferred embodiment the PTH prodrug of the present invention is offormula (IIe-i):

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   -   wherein        -   m and p are independently an integer ranging from and            including 400 to 500.

Preferably, -D is attached to the PTH prodrug of formula (IIe-i) throughthe N-terminal amine functional group of the PTH moiety.

In another preferred embodiment the PTH prodrug of the present inventionis of formula (IIf-i):

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a PTH moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   -   wherein        -   m and p are independently an integer ranging from and            including 400 to 500.

Preferably, -D is attached to the PTH prodrug of formula (IIf-i) throughthe N-terminal amine functional group of the PTH moiety.

In a preferred embodiment the residual activity of the PTH prodrug ofthe present invention is less than 10%, more preferably less than 1%,even more preferably less than 0.1%, even more preferably less than0.01%, even more preferably less than 0.001% and most preferably lessthan 0.0001%.

As used herein the term “residual activity” refers to the activityexhibited by the PTH prodrug of the present invention with the PTHmoiety bound to a carrier in relation to the activity exhibited by thecorresponding free PTH. In this context the term “activity” refers tobinding to an activation of the PTH/PTHrP1 receptor resulting inactivation of adenylate cyclase to generate cAMP, phospholipase C togenerate intracellular calcium, or osteoblastic expression of RANKL(which binds to RANK (Receptor Activator of Nuclear Factor kB) onosteoclasts. It is understood that measuring the residual activity ofthe PTH prodrug of the present invention takes time during which acertain amount of PTH will be released from the PTH prodrug of thepresent invention and that such released PTH will distort the resultsmeasured for the PTH prodrug. It is thus accepted practice to test theresidual activity of a prodrug with a conjugate in which the drugmoiety, in this case PTH, is non-reversibly, i.e. stably, bound to acarrier, which as closely as possible resembles the structure of the PTHprodrug for which residual activity is to be measured.

A suitable assay for measuring PTH activity and the residual activity ofthe PTH prodrug of the present invention, preferably in the form of astable analog, is for example measuring cAMP production from HEK293cells over-expressing the PTH/PTHrP1 receptor (Hohenstein et al.,Journal of Pharmaceutical and Biomedical Analysis, September 2014, 98:345-350), or a cell-based assay to detect cyclicAMP release, detected byhomogenous time-resolved fluorescence (HTRF) or ELISA, that has beenvalidated according to ICHQ2(RT)(http://www.criver.com/files/pdfs/bps/bp_r_in_vitro_bioassays.aspx).

It was surprisingly found that using N-terminal attachment of -L¹- andusing a branched PEG carrier for -Z, i.e. a 2×20 kDa PEG, results in aparticularly low residual activity. Reduced residual activity isdesirable as it reduces side-effects.

It was also surprisingly found that PTH produgs of the present inventionare capable of achieving a stable plasma profile of PTH which ensuresphysiological serum and urinary calcium levels or even reduced urinarycalcium levels.

Preferably, after subcutaneous administration the pharmacokineticprofile of a PTH prodrug of the present invention exhibits a peak totrough ratio of less than 4 within one injection interval.

As used herein the term “injection interval” refers to the time betweentwo consecutive administrations of the pharmaceutical composition of thepresent invention.

As used herein the term “peak to trough ratio” refers to the ratiobetween the highest plasma concentration and the lowest plasmaconcentration of PTH released from the PTH prodrug of the presentinvention within the time period between two consecutive administrationsto a non-human primate, preferably to a cynomolgus monkey.

The time period between two consecutive subcutaneous administrations,i.e. the administration interval, is preferably at least 24 hours, suchas 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, every 84 hours, 96hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, one week,two weeks, three weeks or four weeks.

In one embodiment the time period between two consecutive subcutaneousadministrations is 24 hours.

In another embodiment the time period between two consecutivesubcutaneous administrations is 48 hours.

In another embodiment the time period between two consecutivesubcutaneous administrations is 72 hours.

In another embodiment the time period between two consecutivesubcutaneous administrations is 96 hours.

In another embodiment the time period between two consecutivesubcutaneous administrations is 120 hours.

In another embodiment the time period between two consecutivesubcutaneous administrations is 144 hours.

In another embodiment the time period between two consecutivesubcutaneous administrations is one week.

The peak to trough ratio measured in each administration interval isless than 4, preferably less than 3.8, more preferably less than 3.6,even more preferably less than 3.4, even more preferably less than 3.2,even more preferably less than 3, even more preferably less than 2.8,even more preferably less than 2.6, even more preferably less than 2.4,even more preferably less than 2.2 and most preferably less than 2.

Another aspect of the present invention is a pharmaceutical compositioncomprising at least one PTH prodrug of the present invention and atleast one excipient.

Preferably, the pharmaceutical composition comprising at least one PTHprodrug of the present invention has a pH ranging from and including pH3 to pH 8. More preferably, the pharmaceutical composition has a pHranging from and including pH 4 to pH 6. Most preferably, thepharmaceutical composition has a pH ranging from and including pH 4 topH 5.

In one embodiment the pharmaceutical composition comprising at least onePTH prodrug of the present invention and at least one excipient is aliquid or suspension formulation. It is understood that thepharmaceutical composition is a suspension formulation if the PTHprodrug of the present invention comprises a water-insoluble carrier-Z′.

In another embodiment the pharmaceutical composition comprising at leastone PTH prodrug of the present invention and at least one excipient is adry formulation.

Such liquid, suspension or dry pharmaceutical composition comprises atleast one excipient. Excipients used in parenteral formulations may becategorized as, for example, buffering agents, isotonicity modifiers,preservatives, stabilizers, anti-adsorption agents, oxidation protectionagents, viscosifiers/viscosity enhancing agents, or other auxiliaryagents. However, in some cases, one excipient may have dual or triplefunctions. Preferably, the at least one excipient comprised in thepharmaceutical composition of the present invention is selected from thegroup consisting of

-   (i) Buffering agents: physiologically tolerated buffers to maintain    pH in a desired range, such as sodium phosphate, bicarbonate,    succinate, histidine, citrate and acetate, sulphate, nitrate,    chloride, pyruvate; antacids such as Mg(OH)₂ or ZnCO₃ may be also    used;-   (ii) Isotonicity modifiers: to minimize pain that can result from    cell damage due to osmotic pressure differences at the injection    depot; glycerin and sodium chloride are examples; effective    concentrations can be determined by osmometry using an assumed    osmolality of 285-315 mOsmol/kg for serum;-   (iii) Preservatives and/or antimicrobials: multidose parenteral    formulations require the addition of preservatives at a sufficient    concentration to minimize risk of patients becoming infected upon    injection and corresponding regulatory requirements have been    established; typical preservatives include m-cresol, phenol,    methylparaben, ethylparaben, propylparaben, butylparaben,    chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol,    sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and    benzalkonium chloride;-   (iv) Stabilizers: Stabilisation is achieved by strengthening of the    protein-stabilising forces, by destabilisation of the denatured    state, or by direct binding of excipients to the protein;    stabilizers may be amino acids such as alanine, arginine, aspartic    acid, glycine, histidine, lysine, proline, sugars such as glucose,    sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol,    salts such as potassium phosphate, sodium sulphate, chelating agents    such as EDTA, hexaphosphate, ligands such as divalent metal ions    (zinc, calcium, etc.), other salts or organic molecules such as    phenolic derivatives; in addition, oligomers or polymers such as    cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA    may be used;-   (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or    other proteins or soluble polymers are used to coat or adsorb    competitively to the inner surface of the formulation's container;    e.g., poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35),    polysorbate 20 and 80, dextran, polyethylene glycol,    PEG-polyhistidine, BSA and HSA and gelatins; chosen concentration    and type of excipient depends on the effect to be avoided but    typically a monolayer of surfactant is formed at the interface just    above the CMC value;-   (vi) Oxidation protection agents: antioxidants such as ascorbic    acid, ectoine, methionine, glutathione, monothioglycerol, morin,    polyethylenimine (PEI), propyl gallate, and vitamin E; chelating    agents such as citric acid, EDTA, hexaphosphate, and thioglycolic    acid may also be used;-   (vii) Viscosifiers or viscosity enhancers: in case of a suspension    retard settling of the particles in the vial and syringe and are    used in order to facilitate mixing and resuspension of the particles    and to make the suspension easier to inject (i.e., low force on the    syringe plunger); suitable viscosifiers or viscosity enhancers are,    for example, carbomer viscosifiers like Carbopol 940, Carbopol    Ultrez 10, cellulose derivatives like hydroxypropylmethylcellulose    (hypromellose, HPMC) or diethylaminoethyl cellulose (DEAE or    DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate,    hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans    like Satia gum UTC 30, aliphatic poly(hydroxy acids), such as    poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and    their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and    caprolactone, poloxamers, hydrophilic poly(oxyethylene) blocks and    hydrophobic poly(oxypropylene) blocks to make up a triblock of    poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g.    Pluronic®), polyetherester copolymer, such as a polyethylene glycol    terephthalate/polybutylene terephthalate copolymer, sucrose acetate    isobutyrate (SAIB), dextran or derivatives thereof, combinations of    dextrans and PEG, polydimethylsiloxane, collagen, chitosan,    polyvinyl alcohol (PVA) and derivatives, polyalkylimides, poly    (acrylamide-co-diallyldimethyl ammonium (DADMA)),    polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as    dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin,    heparan sulfate, hyaluronan, ABA triblock or AB block copolymers    composed of hydrophobic A-blocks, such as polylactide (PLA) or    poly(lactide-co-glycolide) (PLGA), and hydrophilic B-blocks, such as    polyethylene glycol (PEG) or polyvinyl pyrrolidone; such block    copolymers as well as the abovementioned poloxamers may exhibit    reverse thermal gelation behavior (fluid state at room temperature    to facilitate administration and gel state above sol-gel transition    temperature at body temperature after injection);-   (viii) Spreading or diffusing agent: modifies the permeability of    connective tissue through the hydrolysis of components of the    extracellular matrix in the intrastitial space such as but not    limited to hyaluronic acid, a polysaccharide found in the    intercellular space of connective tissue; a spreading agent such as    but not limited to hyaluronidase temporarily decreases the viscosity    of the extracellular matrix and promotes diffusion of injected    drugs; and-   (ix) Other auxiliary agents: such as wetting agents, viscosity    modifiers, antibiotics, hyaluronidase; acids and bases such as    hydrochloric acid and sodium hydroxide are auxiliary agents    necessary for pH adjustment during manufacture.

The pharmaceutical composition comprising at least one PTH prodrug maybe administered to a patient by various modes of administration, such asvia topical, enteral or parenteral administration and by methods ofexternal application, injection or infusion, including intraarticular,periarticular, intradermal, subcutaneous, intramuscular, intravenous,intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital,intravitreal, intratympanic, intravesical, intracardiac, transtracheal,subcuticular, subcapsular, subarachnoid, intraspinal, intraventricular,intrasternal injection and infusion, direct delivery to the brain viaimplanted device allowing delivery of the invention or the like to braintissue or brain fluids (e.g., Ommaya Reservoir), directintracerebroventricular injection or infusion, injection or infusioninto brain or brain associated regions, injection into the subchoroidalspace, retro-orbital injection and ocular instillation. Preferably thepharmaceutical composition comprising at least one PTH prodrug isadministered via subcutaneous injection.

Subcutaneous injection is preferably done with a syringe and needle orwith a pen injector, even more preferably with a pen injector.

Another aspect of the present invention is the use of the PTH prodrug ora pharmaceutically acceptable salt thereof or a pharmaceuticalcomposition comprising at least one PTH prodrug of the present inventionas a medicament.

Another aspect of the present invention is the PTH prodrug or apharmaceutically acceptable salt thereof or the pharmaceuticalcomposition comprising at least one PTH prodrug of the present inventionfor use in the treatment of a disease which can be treated with PTH.

Preferably, said disease is selected from the group consisting ofhypoparathyroidism, hyperphosphatemia, osteoporosis, fracture repair,osteomalacia, osteomalacia and osteoporosis in patients withhypophosphatasia, steroid-induced osteoporosis, male osteoporosis,arthritis, osteoarthritis, osteogenesis imperfect, fibrous dysplasia,rheumatoid arthritis, Paget's disease, humoral hypercalcemia associatedwith malignancy, osteopenia, periodontal disease, bone fracture,alopecia, chemotherapy-induced alopecia, and thrombocytopenia. Mostpreferably said disease is hypoparathyroidism.

In one embodiment the patient undergoing the method of treatment of thepresent invention is a mammalian patient, preferably a human patient.

Another aspect of the present invention is the use of the PTH prodrug ora pharmaceutically acceptable salt thereof or the pharmaceuticalcomposition comprising at least one PTH prodrug of the present inventionfor the manufacture of a medicament for treating a disease which can betreated with PTH.

Preferably, said disease is selected from the group consisting ofhypoparathyroidism, hyperphosphatemia, osteoporosis, fracture repair,osteomalacia, osteomalacia and osteoporosis in patients withhypophosphatasia, steroid-induced osteoporosis, male osteoporosis,arthritis, osteoarthritis, osteogenesis imperfect, fibrous dysplasia,rheumatoid arthritis, Paget's disease, humoral hypercalcemia associatedwith malignancy, osteopenia, periodontal disease, bone fracture,alopecia, chemotherapy-induced alopecia, and thrombocytopenia. Mostpreferably said disease is hypoparathyroidism.

In one embodiment the disease to be treated with the PTH prodrug or apharmaceutically acceptable salt thereof or the pharmaceuticalcomposition comprising at least one PTH prodrug of the present inventionoccurs in a mammalian patient, preferably in a human patient.

A further aspect of the present invention is a method of treating,controlling, delaying or preventing in a mammalian patient, preferably ahuman patient, in need of the treatment of one or more diseases whichcan be treated with PTH, comprising the step of administering to saidpatient in need thereof a therapeutically effective amount of PTHprodrug or a pharmaceutically acceptable salt thereof or apharmaceutical composition comprising PTH prodrug of the presentinvention.

Preferably, the one or more diseases which can be treated with PTH isselected from the group consisting of hypoparathyroidism,hyperphosphatemia, osteoporosis, fracture repair, osteomalacia,osteomalacia and osteoporosis in patients with hypophosphatasia,steroid-induced osteoporosis, male osteoporosis, arthritis,osteoarthritis, osteogenesis imperfect, fibrous dysplasia, rheumatoidarthritis, Paget's disease, humoral hypercalcemia associated withmalignancy, osteopenia, periodontal disease, bone fracture, alopecia,chemotherapy-induced alopecia, and thrombocytopenia. Most preferablysaid disease is hypoparathyroidism.

An additional aspect of the present invention is a method ofadministering the PTH prodrug, a pharmaceutically acceptable saltthereof or the pharmaceutical composition of the present invention,wherein the method comprises the step of administering the PTH prodrug,a pharmaceutically acceptable salt thereof or the pharmaceuticalcomposition of the present invention via topical, enteral or parenteraladministration and by methods of external application, injection orinfusion, including intraarticular, periarticular, intradermal,subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal,intrathecal, intracapsular, intraorbital, intravitreal, intratympanic,intravesical, intracardiac, transtracheal, subcuticular, subcapsular,subarachnoid, intraspinal, intraventricular, intrasternal injection andinfusion, intranasal, oral, transpulmonary and transdermaladministration, direct delivery to the brain via implanted deviceallowing delivery of the invention or the like to brain tissue or brainfluids (e.g., Ommaya Reservoir), direct intracerebroventricularinjection or infusion, injection or infusion into brain or brainassociated regions, injection into the subchoroidal space, retro-orbitalinjection and ocular instillation, preferably via subcutaneousinjection.

In a preferred embodiment, the present invention relates to a PTHprodrug or pharmaceutically acceptable salt thereof or a pharmaceuticalcomposition of the present invention, for use in the treatment ofhypoparathyroidism via subcutaneous injection.

EXAMPLES Materials and Methods

Side chain protected PTH(1-34) (SEQ ID NO:51) on TCP resin having Bocprotected N-terminus and ivDde protected side chain of Lys26(synthesized by Fmoc-strategy) was obtained from CASLO ApS, KongensLyngby, Denmark and Peptide Specialty Laboratories GmbH, Heidelberg,Germany.

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus (synthesized by Fmoc-strategy) was obtained from CASLO ApS,Kongens Lyngby, Denmark and Peptide Specialty Laboratories GmbH,Heidelberg, Germany.

PEG 2×20 kDa maleimide, Sunbright GL2-400MA and PEG 2×10 kDa maleimide,Sunbright GL2-200 MA were purchased from NOF Europe N.V., Grobbendonk,Belgium. S-Trityl-6-mercaptohexanoic acid was purchased fromPolypeptide, Strasbourg, France. HATU was obtained from MerckBiosciences GmbH, Schwalbach/Ts, Germany. Fmoc-N-Me-Asp(OBn)-OH wasobtained from Peptide International Inc., Louisville, Ky., USA.Fmoc-Aib-OH was purchased from Iris Biotech GmbH, Marktredwitz, Germany.All other chemicals and reagents were purchased from Sigma Aldrich GmbH,Taufkirchen, Germany, unless a different supplier is mentioned.

Compound 11a (examples 11-15) was synthesized following the proceduredescribed in patent WO29095479A2, example 1.

Syringes equipped with polyethylenene frits (MultiSynTech GmbH, Witten,Germany) were used as reaction vessels or for washing steps of peptideresins.

General procedure for the removal of ivDde protecting group from sidechain protected PTH on resin: The resin was pre-swollen in DMF for 30min and the solvent was discarded. The ivDde group was removed byincubating the resin with DMF/hydrazine hydrate 4/1 (v/v, 2.5 mL/gresin) for 8×15 min. For each step fresh DMF/hydrazine hydrate solutionwas used. Finally, the resin was washed with DMF (10 x), DCM (10 x) anddried in vacuo.

General procedure for the removal of Fmoc protecting group fromprotected PTH on resin: The resin was pre-swollen in DMF for 30 min andthe solvent was discarded. The Fmoc group was removed by incubating theresin with DMF/piperidine/DBU 96/2/2 (v/v/v, 2.5 mL/g resin) for 3×10min. For each step fresh DMF/piperidine/DBU h solution was used.

Finally, the resin was washed with DMF (10 x), DCM (10 x) and dried invacuo.

RP-HPLC Purification:

For preparative RP-HPLC a Waters 600 controller and a 2487 DualAbsorbance Detector was used, equipped with the following columns:Waters XBridge™ BEH300 Prep C18 5 μm, 150×10 mm, flow rate 6 mL/min, orWaters XBridge™ BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 mL/min.Linear gradients of solvent system A (water containing 0.1% TFA v/v) andsolvent system B (acetonitrile containing 0.1% TFA v/v) were used. HPLCfractions containing product were pooled and lyophilized if not statedotherwise.

Flash Chromatography:

Flash chromatography purifications were performed on an Isolera Onesystem from Biotage AB, Sweden, using Biotage KP-Sil silica cartridgesand n-heptane and ethyl acetate as eluents. Products were detected at254 nm.

Ion Exchange Chromatography:

Ion exchange chromatography (IEX) was performed using an AmershamBioscience AEKTAbasic system equipped with a MacroCap SP cationexchanger column (Amersham Bioscience/GE Healthcare). 17 mM acetic acidpH 4.5 (solvent A) and 17 mM acetic acid, 1 M NaCl, pH 4.5 (solvent B)were used as mobile phases.

Size Exclusion Chromatography:

Size exclusion chromatography (SEC) was performed using an AmershamBioscience AEKTAbasic system equipped with HiPrep 26/10 desaltingcolumns (Amersham Bioscience/GE Healthcare). 0.1% (v/v) acetic acid wasused as mobile phase.

For in vitro release kinetics studies of compound 31, a pH 7.40 buffer(100 mM phosphate, 10 mM L-methionine, 3 mM EDTA, 0.05% Tween-20) wasused instead of 0.1% AcOH as mobile phase.

Analytical Methods

Analytical ultra-performance LC (UPLC)-MS was performed on a WatersAcquity system equipped with a Waters BEH300 C18 column (2.1×50 mm, 1.7μm particle size, flow: 0.25 mL/min; solvent A: water containing 0.04%TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v)) coupledto a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific orcoupled to a Waters Micromass ZQ.

Quantitative measurements of serum calcium (sCa), urinary calcium andserum phosporous (sP) were performed on a Roche-Hitachi P800 modularbiochemistry instrument.

Quantification of Plasma Total PTH(1-34) Concentrations:

Plasma total PTH(1-34) concentrations were determined by quantificationof a signature peptide close to the N-terminus (sequence: IQLMHNLGK) anda C-terminal signature peptide (sequence: LQDVHNF) after plasma proteinprecipitation, followed by sequential digestion with EndoproteinaseLys-C (origin: Lysobacter enzymogenes) and Endoproteinase Glu-C (origin:Staphylococcus aureus V8) of the supernatant. Subsequently, analysis byreversed phase liquid chromatography and detection by mass spectrometry(RP-HPLC-MS) was performed.

Calibration standards of PTH(1-34) conjugate in blank plasma wereprepared as follows: The PTH(1-34) conjugate formulation was pre-dilutedwith formulation buffer to aqueous standard solutions ranging from 5 to300 μg/mL PTH(1-34) eq (concentration range 1) and 0.5 μg/mL to 100μg/mL PTH(1-34) eq (concentration range 2), respectively. Each aqueousstandard solution was then diluted 1:100 with thawed heparinized plasmato obtain concentration ranges from 50 to 3000 ng/mL PTH(1-34) eq(dilution with rat plasma of concentration range 1) and 5 to 1000 ng/mLPTH(1-34) eq (dilution with monkey plasma of concentration range 2).

These solutions were used for the generation of a calibration curve.Calibration curves were weighted 1/×2 for both signature peptides. Forquality control, three samples independent from the calibration standardsolutions were prepared accordingly. Concentrations at the lower end(3-5 fold concentration of the respective LLOQ), the middle range(0.05-0.1 fold concentration of the respective ULOQ) and the upper end(0.5-0.8 fold concentration of the respective ULOQ).

Sample preparation volumes can be altered depending on the targetedsignal response after sample preparation. Processing procedure of theprotein precipitation is described here for the analysis of plasmasamples originated in monkey species. Protein precipitation was carriedout by addition of 200 μL of precooled (5-10° C.) methanol to 100 μL ofthe plasma sample. 180 μL of the supernatant were transferred into a newwell-plate and evaporated to dryness (under a gentle nitrogen stream at45° C.). 50 μL of reconstitution solvent (50 mM Tris 0.5 mM CaCl₂buffer, adjusted to pH 8.0) were used to dissolve the residue.Proteolytic digestion was performed as follows:

20 μg of Lys-C (order number 125-05061, Wako Chemicals GmbH, Neuss,Germany) were dissolved in 80 μL of 10 mM acetic acid. 3 μL of the Lys-Csolution were added to each cavity and samples incubated for 15 hours at37° C. Afterwards 10 μg of Glu-C (order number V1651, Promega GmbH,Mannheim, Germany) were dissolved in 25 μL water, added to each cavityand incubation continued for 1.5 hours at 37° C. After incubationsamples were acidified with 2 μL water/formic acid 4:6 (v/v) and 10 μLwere injected into the UPLC-MS system.

LC-MS analysis was carried out by using an Agilent 1290 UPLC coupled toan Agilent 6460 TripleQuad mass spectrometer via an ESI probe.Chromatography was performed on a Waters Acquity BEH300 C18 analyticalcolumn (1.7 μm particle size; column dimensions used are 50×2.1 mm foranalysis of samples originated from rat species or 100×2.1 mm foranalysis of samples originated from monkey species) with pre-filter at aflow rate of 0.30 mL/min (T=60° C.). Water (UPLC grade) containing 0.1%formic acid (v/v) was used as mobile phase A and acetonitrile (UPLCgrade) with 0.1% formic acid as mobile phase B.

The gradient system for the analysis of samples originated from ratplasma comprised a linear increase from 0.1% B to 40% B in 7 min. Thegradient system for the analysis of samples originated from monkeyplasma comprised a linear increase from 8.0% B to 11.0% B in 6 min,followed by a linear increase to 26% B in 4 minutes. Mass analysis wasperformed in multiple reaction monitoring (MRM) mode, monitoring thetransitions m/z 437.2 to 131.0 and m/z 352.3 to 463.0.

Alternatively, quantification of plasma total PTH(1-34) concentrationswas performed according to the following procedure:

Plasma total PTH(1-34) concentrations were determined by quantificationof a signature peptide close to the N-terminus (sequence: IQLMHNLGK) anda C-terminal signature peptide (sequence: LQDVHNF) after plasma proteinprecipitation, followed by sequential digestion with EndoproteinaseLys-C (origin: Lysobacter enzymogenes) and Endoproteinase Glu-C (origin:Staphylococcus aureus V8) of the supernatant. Subsequently, analysis byreversed phase liquid chromatography and detection by mass spectrometry(RP-HPLC-MS) was performed.

Calibration standards of PTH(1-34) conjugate in blank heparinized plasmawere prepared in concentration ranges from 50 to 3000 ng/mL PTH(1-34) eq(dilution with rat plasma) and 1 to 1000 ng/mL PTH(1-34) eq (dilutionwith monkey plasma).

These solutions were used for the generation of a calibration curve. Forquality control, three samples independent from the calibration standardsolutions were prepared accordingly. Concentrations at the lower end(3-5 fold concentration of the respective LLOQ), the middle range(0.05-0.1 fold concentration of the respective ULOQ) and the upper end(0.5-0.8 fold concentration of the respective ULOQ).

Sample preparation volumes can be altered depending on the targetedsignal response after sample preparation. Processing procedure of theprotein precipitation is described here for the analysis of plasmasamples originated in rat species. Protein precipitation was carried outby addition of 100 μL of precooled (5-10° C.) methanol to 50 μL of theplasma sample. 60 μL of the supernatant were transferred into a newwell-plate and evaporated to dryness (under a gentle nitrogen stream at45° C.). 60 μL of reconstitution solvent (50 mM Tris 0.5 mM CaCl₂buffer, adjusted to pH 8.0) were used to dissolve the residue.Proteolytic digestion was performed as follows: 20 μg of Lys-C (ordernumber 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in80 μL of 10 mM acetic acid. 3 μL of the Lys-C solution were added toeach cavity and samples incubated for 15 hours at 37° C. Afterwards 10μg of Glu-C (order number V1651, Promega GmbH, Mannheim, Germany) weredissolved in 25 μL water, and 1.5 μL of the Glu-C solution added to eachcavity and incubation continued for 1.5 hours at 37° C. After incubationsamples were acidified with 2 μL water/formic acid 4:6 (v/v) and 10 μLwere injected into the UPLC-MS system.

Chromatography was performed on a Waters Acquity BEH300 C18 analyticalcolumn (1.7 μm particle size; column dimensions 50×2.1 mm). Water (UPLCgrade) containing 0.1% formic acid (v/v) was used as mobile phase A andacetonitrile (UPLC grade) with 0.1% formic acid as mobile phase B.

Quantification of Plasma PEG Concentrations:

Plasma total PEG concentrations were determined by quantification of thepolymeric part of PTH(1-34) conjugates after plasma proteinprecipitation and enzymatic digestion of the supernatant. Analysis bysize exclusion chromatography and detection by mass spectrometry(SEC-MS) followed.

Calibration standards of PTH(1-34) conjugate in blank heparinized monkeyplasma were prepared in concentration ranges from 50 to 1200 ng/mL PEGequivalents.

These solutions were used for the generation of a quadratic calibrationcurve. Calibration curves were weighted 1/x. For quality control, threesamples independent from the calibration standard solutions wereprepared accordingly. Concentrations at the lower end (2-4 foldconcentration of the LLOQ), the middle range (0.1-0.2 fold concentrationof the ULOQ) and the upper end (0.8 fold concentration of the ULOQ).Protein precipitation was carried out by addition of 200 μL of precooled(5-10° C.) methanol to 100 μL of the plasma sample. 180 μL of thesupernatant were transferred into a new well-plate and evaporated todryness (under a gentle nitrogen stream at 45° C.). 50 μL ofreconstitution solvent (50 mM Tris 0.5 mM CaCl₂ buffer, adjusted to pH8.0) were used to dissolve the residue. Proteolytic digestion wasperformed as follows: 20 μg of Lys-C (order number 125-05061, WakoChemicals GmbH, Neuss, Germany) were dissolved in 80 μL of 10 mM aceticacid. 3 μL of the Lys-C solution were added to each cavity and samplesincubated for 15 hours at 37° C. Afterwards 10 μg of Glu-C (order numberV1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 μL water,and 1.5 μL of the Glu-C solution added to each cavity and incubationcontinued for 1.5 hours at 37° C. After incubation samples wereacidified with 2 μL water/formic acid 4:6 (v/v) and 5 μL were injectedinto the SEC-MS system.

SEC-MS analysis was carried out by using an Agilent 1290 UPLC coupled toan Agilent 6460 TripleQuad mass spectrometer via an ESI probe.Acquisition of a distinct precursor ion of the polymer was achieved byapplying high voltage in-source fragmentation (200-300V) at the MSinterface. Chromatography was performed on a TOSOH TSK Gel SuperAW3000analytical column (4.0 μm particle size; column dimensions 150×6.0 mm)at a flow rate of 0.50 mL/min (T=65° C.). Water (UPLC grade) containing0.1% formic acid (v/v) was used as mobile phase A and acetonitrile (UPLCgrade) with 0.1% formic acid as mobile phase B. The chromatographicsetup for sample analysis comprises an isocratic elution of 50% B over 8minutes.

Mass analysis was performed in single reaction monitoring (SRM) mode,monitoring the transition m/z 133.1 to 45.1.

Due to the reversible nature of the attachment of -L¹- to -D,measurements for PTH receptor activity were made using stable analogs ofthe PTH prodrugs of the present invention, i.e. they were made usingsimilar structures to those of the PTH prodrugs of the presentinvention, which instead of a reversible attachment of -Z to -D have astable attachment.

This was necessary, because the PTH prodrugs of the present inventionwould release PTH in the course of the experiment and said released PTHwould have influenced the result.

Example 1 Synthesis of Linker Reagent 1f

Linker reagent 1f was synthesized according to the following scheme:

To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) andNaCNBH₃ (819 mg, 12.63 mmol) in MeOH (20 mL) was added2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion wise. Themixture was stirred at rt for 90 min, acidified with 3 M HCl (4 mL) andstirred further 15 min. The reaction mixture was added to saturatedNaHCO₃ solution (200 mL) and extracted 5× with DCM. The combined organicphases were dried over Na₂SO₄ and the solvents were evaporated in vacuo.The resulting N-methyl-N-Boc-N′-Tmob-ethylenediamine 1a was dried inhigh vacuum and used in the next reaction step without furtherpurification.

-   Yield: 3.76 g (11.48 mmol, 89% purity, 1a: double Tmob protected    product=8:1)-   MS: m/z 355.22=[M+H]⁺, (calculated monoisotopic mass=354.21).

To a solution of 1a (2 g, 5.65 mmol) in DCM (24 mL) COMU (4.84 g, 11.3mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine(2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3h at rt, diluted with DCM (250 mL) and washed 3×with 0.1 M H₂SO₄ (100mL) and 3× with brine (100 mL). The aqueous phases were re-extractedwith DCM (100 mL). The combined organic phases were dried over Na₂SO₄,filtrated and the residue concentrated to a volume of 24 mL. 1b waspurified using flash chromatography.

-   Yield: 5.31 g (148%, 6.66 mmol)-   MS: m/z 796.38=[M+H]+, (calculated monoisotopic mass=795.37).

To a solution of 1b (5.31 g, max. 4.52 mmol ref. toN-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) DBU (1.8 mL, 3% v/v) was added.The solution was stirred for 12 min at rt, diluted with DCM (400 mL) andwashed 3× with 0.1 M H₂SO₄ (150 mL) and 3× with brine (150 mL). Theaqueous phases were re-extracted with DCM (100 mL). The combined organicphases were dried over Na₂SO₄ and filtrated. 1c was isolated uponevaporation of the solvent and used in the next reaction without furtherpurification.

-   MS: m/z 574.31=[M+H]+, (calculated monoisotopic mass=573.30).

1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) andCOMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reactionmixture was stirred for 4 h at rt, diluted with DCM (400 mL) and washed3× with 0.1 M H₂SO₄ (100 mL) and 3× with brine (100 mL). The aqueousphases were re-extracted with DCM (100 mL). The combined organic phaseswere dried over Na₂SO₄, filtered and 1d was isolated upon evaporation ofthe solvent. Product 1d was purified using flash chromatography.

-   Yield: 2.63 g (6200 94% purity)-   MS: m/z 856.41=[M+H]+, (calculated monoisotopic mass=855.41).

To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H₂O (11mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture wasstirred for 70 min at rt. The mixture was diluted with DCM (200 mL) andwashed 3× with 0.1 M H₂SO₄ (50 mL) and 3× with brine (50 mL). Theaqueous phases were re-extracted with DCM (100 mL). The combined organicphases were dried over Na₂SO₄, filtered and 1e was isolated uponevaporation of the solvent. 1e was purified using flash chromatography.

-   Yield: 2.1 g (88%)-   MS: m/z 878.4=[M+Na]⁺, (calculated monoisotopic mass=837.40).

To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) wereadded DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5min, N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and thereaction mixture was stirred at rt for 1 h. The reaction mixture wasfiltered, the solvent was removed in vacuo and the residue was taken upin 90% acetonitrile plus 0.1% TFA (3.4 mL). The crude mixture waspurified by RP-HPLC. Product fractions were neutralized with 0.5 M pH7.4 phosphate buffer and concentrated. The remaining aqueous phase wasextracted with DCM and if was isolated upon evaporation of the solvent.

-   Yield: 154 mg (81%)-   MS: m/z 953.4=[M+H]⁺, (calculated monoisotopic mass=952.43).

Example 2 Synthesis of Linker Reagent 2g

4-Methoxytriphenylmethyl chloride (3.00 g, 9.71 mmol) was dissolved inDCM (20 mL) and added dropwise under stirring to a solution ofethylenediamine 2a (6.5 mL, 97.3 mmol) in DCM (20 mL). The reactionmixture was stirred for 2 h at rt after which it was diluted withdiethyl ether (300 mL), washed 3× with brine/0.1 M NaOH 30/1 (v/v) andonce with brine. The organic phase was dried over Na₂SO₄ and 2b wasisolated upon evaporation of the solvent.

-   Yield: 3.18 g (98%)

Mmt protected intermediate 2b (3.18 g, 9.56 mmol) was dissolved in DCM(30 mL). 6-(Tritylthio)-hexanoic acid (4.48 g, 11.5 mmol), PyBOP (5.67g, 10.9 mmol) and DIPEA (5.0 mL, 28.6 mmol) were added and the mixturewas stirred for 30 min at rt. The solution was diluted with diethylether (250 mL), washed 3× with brine/0.1 M NaOH 30/1 (v/v) and once withbrine. The organic phase was dried over Na₂SO₄ and the solvent wasremoved in vacuo. 2c was purified using flash chromatography.

-   Yield: 5.69 g (85%)-   MS: m/z 705.4=[M+H]⁺, (calculated monoisotopic mass=704.34).

Compound 2c (3.19 g, 4.53 mmol) was dissolved in abhydrous THF (50 mL),1 M BH₃·THF solution in THF (8.5 mL, 8.5 mmol) was added and the mixturewas stirred for 16 h at rt. More 1 M BH₃·THF solution in THF (14 mL,14.0 mmol) was added and the mixture was stirred for further 16 h at rt.Methanol (8.5 mL) and N,N′-dimethyl-ethylendiamine (3.00 mL, 27.9 mmol)were added and the mixture was heated under reflux for 3 h. The mixturewas allowed to cool down and ethyl acetate (300 mL) was added. Thesolution was washed 2× with aqueous Na₂CO₃ and 2× with aqueous NaHCO₃.The organic phase was dried over Na₂SO₄ and the solvent was removed invacuo to obtain 2d.

-   Yield: 3.22 g (103%)-   MS: m/z 691.4=[M+H]⁺, (calculated monoisotopic mass=690.36).

Di-tert-butyl dicarbonate (2.32 g, 10.6 mmol) and DIPEA (3.09 mL, 17.7mmol) were dissolved in DCM (5 mL) and added to a solution of 2d (2.45g, 3.55 mmol) in DCM (5 mL). The mixture was stirred for 30 min at rt.The solution was concentrated in vacuo and purified by flashchromatography to obtain product 2e.

-   Yield: 2.09 g (74%)-   MS: m/z 791.4=[M+H]⁺, (calculated monoisotopic mass=790.42).

Compound 2e (5.01 g, 6.34 mmol) was dissolved in acetonitrile (80 mL).0.4 M aqueous HCl (80 mL) followed by acetonitrile (20 mL) was added andthe mixture was stirred for 1 h at rt.

The pH was adjusted to pH 5.5 by addition of aqueous 5 M NaOH. Theorganic solvent was removed in vacuo and the remaining aqueous solutionwas extracted 4× with DCM. The combined organic phases were dried overNa₂SO₄ and the solvent was removed in vacuo to obtain product 2f.

-   Yield: 4.77 g (95%)-   MS: m/z 519.3=[M+H]⁺, (calculated monoisotopic mass=518.30).

Compound 2f (5.27 g, 6.65 mmol) was dissolved in DCM (30 mL) and addedto a solution of p-nitrophenyl chloroformate (2.01 g, 9.98 mmol) in DCM(25 mL). 2,4,6-trimethylpyridine (4.38 mL, 33.3 mmol) was added and thesolution was stirred for 45 min at rt. The solution was concentrated invacu and purified by flash chromatography to obtain product 2g.

-   Yield: 4.04 g (89%)-   MS: m/z 706.32=[M+Na]⁺, (calculated monoisotopic mass=683.30).

Example 3 Synthesis of Permanent S1 PTH(1-34) Conjugate 3

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of 6-tritylmercaptohexanoic acid (62.5mg, 160 μmol), PyBOP (80.1 mg, 154 μmol) DIPEA (53 μL, 306 μmol) in DMF(2 mL) was added to 0.21 g (51 μmol) of the resin. The suspension wasagitated for 80 min at rt. The resin was washed 10× with DMF, 10× withDCM and dried in vacuo. Cleavage of the peptide from the resin andremoval of protecting groups was achieved by adding 10 mL cleavagecocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole andagitating the suspension for 1 h at rt. Crude 3 was precipitated inpre-cooled diethyl ether (−18° C.). The precipitate was dissolved inACN/water and purified by RP-HPLC. The product fractions werefreeze-dried.

-   Yield: 36 mg (14%), 3*8 TFA-   MS: m/z 1062.31=[M+4H]⁴⁺, (calculated monoisotopic mass for    [M+4H]⁴⁺=1062.30).

Example 4 Synthesis of Permanent K26 PTH(1-34) Conjugate 4

Side chain protected PTH(1-34) on TCP resin having Boc protectedN-terminus and ivDde protected side chain of Lys26 was ivDde deprotectedaccording to the procedure given in Materials and Methods. A solution of6-tritylmercaptohexanoic acid (107 mg, 273 μmol), PyBOP (141 mg, 273μmol) and DIPEA (95 μL, 545 μmol) in DMF (3 mL) was added to 0.80 g(90.9 μmol) of the resin. The suspension was agitated for 1 h at rt. Theresin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavageof the peptide from the resin and removal of protecting groups wasachieved by adding 6 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h atrt. Crude 4 was precipitated in pre-cooled diethyl ether (−18° C.). Theprecipitate was dissolved in ACN/water and purified by RP-HPLC. Theproduct fractions were freeze-dried.

-   Yield: 40 mg (8%), 4*8 TFA-   MS: m/z 1062.30=[M+4H]⁴, (calculated monoisotopic mass for    [M+4H]⁴+=1062.30).

Example 5 Synthesis of Transient S1 PTH(1-34) Conjugate

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of Fmoc-Aib-OH (79 mg, 244 μmol),PyBOP (127 mg, 244 μmol) and DIPEA (64 μL, 365 μmol) in DMF (1.5 mL) wasadded to 0.60 g (61 μmol) of the resin. The suspension was agitated for16 h at rt. The resin was washed 10× with DMF and Fmoc-deprotected asdescribed above. A solution of 2g (167 mg, 244 μmol) and DIPEA (64 μL,365 μmol) in DMF (1.5 mL) was added to the resin. The suspension wasagitated for 24 h at rt. The resin was washed 10× with DMF, 10× with DCMand dried in vacuo. Cleavage of the peptide from the resin and removalof protecting groups was achieved by adding 7 mL cleavage cocktail100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating thesuspension for 1 h at rt. Crude 5 was precipitated in pre-cooled diethylether (−18° C.). The precipitate was dissolved in ACN/water and purifiedby RP-HPLC. The product fractions were freeze-dried.

-   Yield: 78 mg (24%), 5*9 TFA-   MS: m/z 1101.59=[M+4H]⁴⁺, (calculated monoisotopic mass for    [M+4H]⁴⁺=1101.57).

Example 6 Synthesis of Transient S1 PTH(1-34) Conjugate 6

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of Fmoc-Ala-OH (32 mg, 102 μmol),PyBOP (53 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) wasadded to 0.25 g (25 μmol) of the resin. The suspension was shaken for 1h at rt. The resin was washed 10× with DMF, 10× with DCM and dried undervacuum. Fmoc-deprotection was performed as described above. A solutionof 2g (69 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) wasadded to the resin. The suspension was agitated for 1.5 h at rt. Theresin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavageof the peptide from the resin and removal of protecting groups wasachieved by adding 3 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h atrt. Crude 6 was precipitated in pre-cooled diethyl ether (−18° C.). Theprecipitate was dissolved in ACN/water and purified by RP-HPLC. Theproduct fractions were freeze-dried.

-   Yield: 25 mg (18%), 6*9 TFA-   MS: m/z 1098.75=[M+4H]⁴⁺, (calculated monoisotopic mass for    [M+4H]⁴⁺=1098.07).

Example 7 Synthesis of Transient S1 PTH(1-34) Conjugate 7

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of Fmoc-Ser (Trt)-OH (117 mg, 205μmol), PyBOP (108 mg, 207 μmol) and DIPEA (53 μL, 305 μmol) in DMF (2mL) was added to 0.50 g (51 μmol) of the resin. The suspension wasagitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCMand dried under vacuum. Fmoc-deprotection was performed as describedabove. A solution of 2g (144 mg, 211 μmol) and DIPEA (53 μL, 305 μmol)in DMF (1.8 mL) was added to the resin. The suspension was shaken for 7h at rt. The resin was washed 10× with DMF, 10× with DCM and dried invacuo. Cleavage of the peptide from the resin and removal of protectinggroups was achieved by adding 6 mL cleavage cocktail 100/3/3/2/1(v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspensionfor 1 h at rt. Crude 7 was precipitated in pre-cooled diethyl ether(−18° C.). The precipitate was dissolved in ACN/water and purified byRP-HPLC. The product fractions were freeze-dried.

-   Yield: 54 mg (20%), 7*9 TFA-   MS: m/z 1102.08=[M+4H]⁴, (calculated monoisotopic mass for    [M+4H]⁴+=1102.07).

Example 8 Synthesis of Transient S1 PTH(1-34) Conjugate 8

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of Fmoc-Leu-OH (36 mg, 102 μmol),PyBOP (53 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) wasadded to 0.25 g (25 μmol) of the resin. The suspension was agitated for1 h at rt. The resin was washed 10× with DMF, 10× with DCM and driedunder vacuum. Fmoc-deprotection was performed as described above. Asolution of 2g (69 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3mL) was added to the resin. The suspension was agitated for 1.5 h at rt.The resin was washed 10× with DMF, 10× with DCM and dried in vacuo.Cleavage of the peptide from the resin and removal of protecting groupswas achieved by adding 3 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h atrt. Crude 8 was precipitated in pre-cooled diethyl ether (−18° C.). Theprecipitate was dissolved in ACN/water and purified by RP-HPLC. Theproduct fractions were freeze-dried.

-   Yield: 31 mg (22%), 8*9 TFA-   MS: m/z 1109.32=[M+4H]⁴⁺, (calculated monoisotopic mass for    [M+4H]⁴+=1108.58).

Example 9 Synthesis of Transient S1 PTH(1-34) Conjugate 9

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of 1e (182 mg, 213 μmol), PyBOP (111mg, 213 μmol) and DIPEA (93 μL, 532 μmol) in DMF (5 mL) was added to2.00 g (107 μmol) of the resin. The suspension was agitated for 16 h atrt. The resin was washed 10× with DMF, 10× with DCM and dried undervacuum. Cleavage of the peptide from the resin and removal of protectinggroups was achieved by adding 20 mL cleavage cocktail 100/3/3/2/1(v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspensionfor 1 h at rt. Crude 9 was precipitated in pre-cooled diethyl ether(−18° C.). The precipitate was dissolved in ACN/water and purified byRP-HPLC. The product fractions were freeze-dried.

-   Yield: 47 mg (8%), 9*9 TFA-   MS: m/z 1108.58=[M+4H]⁴⁺, (calculated monoisotopic mass for    [M+4H]⁴+1108.57).

Example 10 Synthesis of Transient K26 PTH(1-34) Conjugate 10

Side chain protected PTH(1-34) on TCP resin having Boc protectedN-terminus and ivDde protected side chain of Lys26 was ivDde deprotectedaccording to the procedure given in Materials and Methods. A solution ofif (867 mg, 910 μmol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) wasadded to 1.91 g (227 μmol) of the resin. The suspension was agitated for1 h at rt. The resin was washed 10× with DMF, 10× with DCM and driedunder vacuum. Cleavage of the peptide from the resin and removal ofprotecting groups was achieved by adding 20 mL cleavage cocktail100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and shaking thesuspension for 1 h at rt. Crude 10 was precipitated in pre-cooleddiethyl ether (−18° C.). The precipitate was dissolved in ACN/water andpurified by RP-HPLC. The product fractions were freeze-dried.

-   Yield: 92 mg (7%), 10*9 TFA-   MS: m/z 1108.58=[M+4H]⁴⁺, (calculated monoisotopic mass for    [M+4H]⁴+1108.57).

Example 11 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate11b

0.15 mL of a 0.5 M NaH₂PO₄ buffer (pH 7.4) was added to 0.5 mL of a 20mg/mL solution of thiol 5 (10 mg, 1.84 μmol) in 1/1 (v/v)acetonitrile/water containing 0.1% TFA (v/v). The solution was incubatedat rt for 10 min after which 238 μL of a 10 mg/mL solution of maleimide11a (2.4 mg, 2.21 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1%TFA (v/v) were added. The solution was incubated for 20 min at rt. 10 μLTFA was added and the mixture was purified by RP-HPLC. The productfractions were freeze-dried to obtain 1b.

-   Yield: 3.1 mg (26%), 11b*9 TFA-   MS: m/z 1097.00=[M+4H]⁴, (calculated monoisotopic mass for    [M+5H]⁵⁺=1096.99).

Example 12 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate12

Conjugate 12 was synthesized as described for 1b by using thiol 6 (10mg, 1.85 μmol) and maleimide 11a (2.4 mg, 2.21 μmol).

-   Yield: 10 mg (83%), 12*9 TFA-   MS: m/z 1094.20=[M+4H]⁴, (calculated monoisotopic mass for    [M+4H]⁴+=1094.19).

Example 13 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate13

Conjugate 13 was synthesized as described for 1b by using thiol 7 (10mg, 1.84 μmol) and maleimide 11a (2.4 mg, 2.21 μmol).

-   Yield: 8 mg (67%), 13*9 TFA-   MS: m/z 1097.40=[M+5H]⁵⁺, (calculated monoisotopic mass for    [M+5H]⁵⁺=1097.39).

Example 14 Synthesis of Low Molecular Weight Transient S1 PEG Conjugate14

Conjugate 14 was synthesized as described for 1b by using thiol 8 (10mg, 1.83 μmol) and maleimide 11a (2.4 mg, 2.21 μmol).

-   Yield: 4 mg (33%), 14*9 TFA-   MS: m/z 1378.01=[M+4H]⁴, (calculated monoisotopic mass for    [M+4H]⁴+=1378.00).

Example 15 Synthesis of Low Molecular Weight Transient K26 PEG Conjugate15

Conjugate 15 was synthesized as described for 1b by using thiol 10 (5.2mg, 0.95 μmol) and maleimide 11a (1.23 mg, 1.14 μmol).

-   Yield: 2.1 mg (33%), 15*9 TFA-   MS: m/z 1102.60=[M+5H]⁵⁺, (calculated monoisotopic mass for    [M+5H]5+=1102.59).

Example 16 Synthesis of Permanent 2×20 kDa S1 PEG Conjugate 16

772 μL of a solution containing thiol 3 (19.4 mg/mL, 15 mg, 3.54 μmol)and 2.5 mg/mL Boc-L-Met in 1/1 (v/v) acetonitrile/water containing 0.1%TFA (v/v) were added to 1.87 mL of a solution containing PEG 2×20 kDamaleimide (Sunbright GL2-400MA, 187 mg, 4.32 μmol) and 2.5 mg/mLBoc-L-Met in water containing 0.1% TFA (v/v). 0.5 M NaH₂PO₄ buffer (0.66mL, pH 7.0) was added and the mixture was stirred for 30 min at rt. 10μL of a 270 mg/mL solution of 2-mercaptoethanol in water was added. Themixture was stirred for 5 min at rt and 0.33 mL 1 M HCl were added.Conjugate 16 was purified by IEX followed by RP-HPLC using a lineargradient of solvent system A (water containing 0.1% AcOH v/v) andsolvent system B (acetonitrile containing 0.1% AcOH v/v). The productcontaining fractions were freeze-dried.

-   Yield: 97 mg (2.01 μmol, 57%) conjugate 16*8 AcOH

Example 17 Synthesis of Permanent 2×20 kDa K26 PEG Conjugate 17

Conjugate 17 was prepared as described for 16 by reaction of thiol 4 (15mg, 3.53 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 187 mg,4.32 μmol).

-   Yield: 80 mg (1.79 μmol, 51%) conjugate 17*8 AcOH

Example 18 Synthesis of Transient 2×20 kDa S1 PEG Conjugate 18

Conjugate 18 was prepared as described for 16 by reaction of thiol 5 (37mg, 8.40 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 445 mg,9.24 μmol). The reaction was quenched by addition of 50 μL TFA withoutprior addition of 2-mercaptoethanol. Conjugate 18 was purified by IEXfollowed by SEC for desalting. The product containing fractions werefreeze-dried.

-   Yield: 161 mg (3.33 μmol, 40%) conjugate 18*9 AcOH

Example 19 Synthesis of Transient 2×20 kDa S1 PEG Conjugate 19

Conjugate 19 was prepared as described for 16 by reaction of thiol 7 (27mg, 6.14 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 325 mg,7.50 μmol).

-   Yield: 249 mg (5.16 μmol, 84%) conjugate 19*9 AcOH

Example 20 Synthesis of Transient 2×20 kDa S1 PEG Conjugate 20

Conjugate 20 was prepared as described for 16 by reaction of thiol 9 (38mg, 8.59 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 455 mg,9.45 μmol). The reaction was quenched by addition of 50 μL TFA withoutprior addition of 2-mercaptoethanol. Conjugate 20 was purified by IEXfollowed by SEC for desalting. The product containing fractions werefreeze-dried.

-   Yield: 194 mg (4.01 μmol, 47%) conjugate 20*9 AcOH

Example 21 Synthesis of Transient 2×20 kDa K26 PEG Conjugate 21

Conjugate 21 was prepared as described for 16 by reaction of thiol 10(34 mg, 7.58 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 401mg, 9.26 μmol).

-   Yield: 256 mg (5.30 μmol, 70%) conjugate 21*9 AcOH

Example 22

In Vitro Release Kinetics of Transient Low Molecular Weight PEGConjugates Conjugates 11b, 12, 13, 14, and 15 were dissolved in pH 7.4phosphate buffer (60 mM NaH₂PO₄, 3 mM EDTA, 0.01% Tween-20, adjusted topH 7.4 by NaOH) containing 0.05 mg/mL pentafluorophenol as internalstandard at a concentration of approximately 1 mg conjugate/mL. Thesolutions were filtered sterile and incubated at 37° C. At time points,aliquots were withdrawn and analysed by RP-HPLC and ESI-MS. The fractionof released PTH at a particular time point was calculated from the ratioof UV peak areas of liberated PTH and PEG conjugate. The % released PTHwas plotted against incubation time. Curve-fitting software was appliedto calculate the corresponding half times of release.

Results:

For conjugate 11b a release half life time of 3.2 d was obtained.

For conjugate 12 a release half life time of 8.7 d was obtained.

For conjugate 13 a release half life time of 10.8 d was obtained.

For conjugate 14 a release half life time of 25.3 d was obtained.

For conjugate 15 a release half life time of 6.9 d was obtained.

Example 23 In Vitro Release Kinetics of Transient 2×20 kDa PEGConjugates

Conjugates 18, 19, 20, and 21 were dissolved in pH 7.4 phosphate buffer(60 mM NaH₂PO₄, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 by NaOH)containing 0.08 mg/mL pentafluorophenol as internal standard at aconcentration of approximately 5 mg conjugate/mL. The solutions werefiltered sterile and incubated at 37° C. At time points, aliquots werewithdrawn and analysed by RP-HPLC. The fraction of released PTH at aparticular time point was calculated from the ratio of UV peak areas ofliberated PTH and PEG conjugate. The % released PTH was plotted againstincubation time. Curve-fitting software was applied to calculate thecorresponding half times of release.

Results:

For conjugate 18 a release half life time of 2.8 d was obtained.

For conjugate 19 a release half life time of 13.4 d was obtained.

For conjugate 20 a release half life time of 1.3 d was obtained.

For conjugate 21 a release half life time of 7.1 d was obtained.

Example 24 PTH Receptor Activity of Permanent 2×20 kDa PEG Conjugates 16and 17 in Cell Based Assay

The residual PTH activity of permanently PEGylated conjugates 16 and 17was quantified by measuring cAMP production from HEK293 cellsover-expressing the PTH/PTHrP1 receptor (Hohenstein A, Hebell M, ZikryH, El Ghazaly M, Mueller F, Rohde, J. Development and validation of anovel cell-based assay for potency determination of human parathyroidhormone (PTH), Journal of Pharmaceutical and Biomedical AnalysisSeptember 2014, 98: 345-350). PTH(1-34) from NIBSC (National Institutefor Biological Standards and Control, UK) was used as referencestandard.

Results:

For conjugate 16 a receptor activity of 0.12% was found relative toPTH(1-34) reference.

For conjugate 17 a receptor activity of 0.11% was found relative toPTH(1-34) reference.

The results indicate an effective lowering of receptor activity in thepermanent 2×20 kDa PEG conjugates 16 and 17. It can be concluded thatsimilar conjugates with transiently Ser1 or Lys26 linked PTH (like e.g.18 and 21) are suitable PTH prodrugs providing low residual receptoractivity. Direct analysis of transient conjugates in the cell assay isnot possible due to linker cleavage under the assay conditions. Thereleased PTH would influence the assay result.

Example 25 Pharmacokinetic Study of Permanent 2×20 kDa PEG Conjugates 16and 17 in Rats

Male Wistar rats (6 weeks, 230-260 g) received either a singleintravenous (2 groups, n=3 animals each) or a single subcutaneous (2groups, n=3 animals each) administration of 16 or 17 at doses of 29μg/rat PTH_(eq) and 31 μg/rat PTH_(eq) respectively. Blood samples werecollected up to 168 h post dose, and plasma was generated. PlasmaPTH(1-34) concentrations were determined by quantification of theN-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminalsignature peptide (sequence: LQDVHNF) after LysC and GluC digestion asdescribed in Materials and Methods.

Results: Dose administrations were well tolerated with no visible signsof discomfort during administration and following administration. Nodose site reactions were observed any time throughout the study. Afterintraveneous injection of 16 and 17 the total PTH(1-34) t_(max) wasobserved at 15 min (earliest time point analyzed), followed by a slowdecay in total PTH(1-34) content with a half life time of approx. 13 hand 11 h respectively. After subcutaneous injection the total PTH(1-34)concentration peaked at a t_(m)ax of 24 h for both 16 and 17, followedby a slow decay in total PTH(1-34) content with half life times ofapprox. 1.5 days for both conjugates. The bioavailability was approx.40% and 60% respectively. Similar PK curves were obtained for the N— andthe C-terminal signature peptide up to 168 h post dose, indicating thepresence of intact PTH(1-34) in the conjugate.

The favourable long lasting PK and the stability of PTH in theconjugates indicate the suitability of the permanent 2×20 kDa PEG modelcompounds as slow releasing PTH prodrugs after subcutaneous injection.It can be concluded that similar conjugates with transiently Ser1 (likee.g. 18) or Lys26 linked PTH are suitable PTH prodrugs providing longlasting levels of released bioactive PTH.

Example 26 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate19 in Cynomolgus Monkeys

Male non naïve cynomolgus monkeys (2-4 years, 3.7-5.4 kg) received asingle subcutaneous (n=3 animals) administration of 19 at a dose of 70μg/kg PTH_(eq). Blood samples were collected up to 504 h post dose, andplasma was generated. Total plasma PTH(1-34) concentrations weredetermined by quantification of the N-terminal signature peptide(sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence:LQDVHNF) after LysC and GluC digestion as described in Materials andMethods. The PEG concentrations were determined using the methoddescribed in Materials and Methods.

Results: Dose administrations were well tolerated with no visible signsof discomfort during administration. One animal showed visible signs ofdiscomfort 72 h post dose, but recovered the days after. No dose sitereactions were observed any time throughout the study. The totalPTH(1-34) concentration peaked at a t_(max) of 24 h, followed by a slowdecay in total PTH(1-34) content with a half life time of approx. 2.5 dfor the N-terminal signature peptide and 0.9 d for the C-terminalsignature peptide. The PEG concentration peaked at t_(max) of 24 h,followed by a slow decay in PEG concentration with a half life time of3.5 d.

It can be concluded that conjugate 19 is a suitable prodrug forsustained delivery of PTH.

Example 27 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate18 in Cynomolgus Monkeys

Non naïve cynomolgus monkeys (2-3 years, 2.5-4 kg) received dailysubcutaneous (n=2 animals—1 male/1 female) administration of 18 at doselevels of 0.2, 0.5, and 1 μg/kg PTH_(eq) for 28 days. Blood samples werecollected up to 28 days (at days 1, 13, and, 27 samples were collectedat pre-dose, 2 h, 4 h, 8 h, 12 h, and 24 h post-dose) and plasma wasgenerated. Plasma PTH(1-34) concentrations were determined byquantification of the N-terminal signature peptide (sequence: IQLMHNLGK)and the C-terminal signature peptide (sequence: LQDVHNF) after LysC andGluC digestion as described in Materials and Methods.

Results: All dose administrations were performed without incident. Nodose site reactions were observed any time throughout the study. Doselinearity was observed in the three groups. Dose stacking was observedfrom day 1 compared with day 13 and day 27. Total PTH(1-34)concentrations were quantified via the N-terminal signature peptide(sequence: IQLMHNLGK) at steady state (during day 27).

A low peak-to-trough ratio of total PTH(1-34) for all dose groups ofbelow 3 was observed after daily subcutaneous application at steadystate in cynomolgus monkeys. As free peptide concentrations at steadystate are correlated to total PTH(1-34) concentration, thepeak-to-trough ratio for the free peptide is below 4 in cynomolgusmonkeys.

Example 28

Pharmacodynamic Actions in Thyroparathyroidectomised (TPTx) Rats Duringa 28-Days Study with Daily Subcutaneous Injections with Conjugate 18 orPTH(1-84)

This study was performed in order to test and compare the effect ofdaily subcutaneous injection of compound 18 and PTH(1-84), the currentstandard of care, in an animal disease model relevant for investigatingtreatment of hypoparathyroidism (HP). Rats subjected tothyroparathyroidectomy (TPTx) by blunt dissection are unable to produceparathyroid hormone, PTH, the major regulator of calcium homeostasis.Hence, TPTx rats develop hypocalcemia and hyperphosphatemiacharacteristic of HP. 17 weeks old female SD TPTx rats (n=9/group) weredosed subcutaneously for 28 days with compound 18 (5 μg PTH eq/kg/d; 1.2nmol/kg/d, in 10 mM succinic acid, 46 g/L mannitol, pH 4.0), PTH(1-84)(70 μg PTH eq/kg/d; 7.3 nmol/kg/d; in 10 mM citrate, mannitol 39.0 g/L,pH 5.0) or vehicle.

Additionally, one group of sham operated rats (n=9) representingnormophysiological background control were also given vehicle. Serumcalcium (sCa) and phosporous (sP) levels in the animals were measuredpre- and post-dose on days 1, 6, 12 and 27. Moreover, bone turnovermarkers (P1NP and CTx) were measured and bone quality assessed by exvivo pQCT.

Results: The average sCa in the TPTx rats pre-dosing at day 1 was 8.3mg/dL compared to 10.9 mg/dL in the sham operated control rats. The sPvalues were 8.7 mg/dL and 5.9 mg/dL, respectively. Compound 18 givendaily at 1.2 nmol/kg elevated sCa to near-normal levels while loweringsP within a few days of administration. At day 12 (day 5 at steady statewith compound 18) sCa had stabilised at normal level (10.7 mg/dL) inthis group of animals (compound 18/sham-control ratio=1.01) as opposedto the hypocalceamic level (8.1 mg/dL) measured in the PTH(1-84) treatedrats (PTH(1-84)/sham-control ratio=0.76). Additionally, the 24-hoururinary Ca excretion at day 12 was comparable between the animalstreated with compound 18 and sham-control. Bone mineral density (BMD)and bone mineral content (BMC) were increased in TPTx controls as seenin HP patients. Treatment with Compound 18 decreased BMD, BMC and areain parallel with an increase in CTx compared to sham and vehicle-treatedTPTx animals. A significant increase in trabecular BMD was observed inanimals dosed with PTH(1-84) compared to both control groups.

It was concluded that compound 18 at a dosage even as low as less than20% of the molar equivalent of the here tested dose of PTH(1-84) wasable to maintain sCa at a level comparable to the sCa level insham-control animals (here representing normal level) over a 24 hourperiod. In contrast, PTH(1-84) at a dose of 7.3 nmol/kg/d did not leadto increase in sCa as compared to the levels in the vehicle-injectedTPTx rats. However, a minimal decrease in sP was observed in thePTH(1-84) dosed animals confirming exposure and response to PTH(1-84) inthe rats. Following the 28-days of treatment with compound 18,trabecular and cortical BMD in vertebrae were within normal range,whereas an anabolic effect was observed for PTH(1-84) on trabecular andcortical bone in vertebrae.

Example 29 Synthesis of Linker Reagent 29h

To a solution of compound 29a (250 g, 294 mmol, 1 eq) in dichloromethane(1 L) was added a solution of Na₂CO₃ (187 g, 1.8 mol, 6 eq) in H₂O (1L). The reaction solution was stirred at 15-30° C. for 0.5 hour. TLC(DCM/MeOH=10:1, R_(f)=0.5) showed the starting material was consumedcompletely. The organic layer was separated and the aqueous phase wasextracted with dichloromethane (1 L). The organic layers were combinedand washed with brine (800 mL), then dried with anhydrous Na₂SO₄,filtered and concentrated in vacuum to give compound 29b as a yellowoil.

-   Yield: 200 g, 272 mmol, 93%

Four reactions were carried out in parallel.

To a solution of compound 29b (50 g, 68.1 mmol, 1 eq) andFmoc-5-aminovaleric acid (25.4 g, 74.9 mmol, 1.1 eq), DIPEA (61.6 g, 477mmol, 83.3 mL, 7 eq) in acetonitrile (500 mL) was added drop-wise T3P50% [EtOAc] (130 g, 204 mmol, 122 mL, 3 eq) at 15-30° C. for 1 hour.After addition, the reaction mixture was stirred at 15-30° C. for 18hours. TLC (Petroleum ether/Ethyl acetate=1:1, R_(f)=0.5) showed thestarting material was consumed completely. The four reactions werecombined for workup. The mixture was diluted with water (3 L), thenadjusted to pH=3˜4 with 0.5 N HCl solution. The mixture was extractedwith EtOAc (3 L), then the aqueous phase was extracted with EtOAc (2 L).The organic layers were combined and washed with brine (1 L), then driedwith anhydrous Na₂SO₄, filtered and concentrated in vacuum to give thecrude product as yellow oil. The crude product was purified by columnchromatography on silica gel with petroleum ether/ethyl acetate to givecompound 29c as a yellow solid.

-   Yield: 220 g, 199 mmol, 73%

Four reactions were carried out in parallel.

To a solution of compound 29c (55 g, 52 mmol, 1 eq) in dichloromethane(275 mL) was added piperidine (47.3 g, 555 mmol, 55 mL, 10.7 eq). Thereaction solution was stirred for 3 hours at 15-30° C. TLC (Petroleumether/Ethyl acetate=1:1, R_(f)=0) showed the starting material wasconsumed completely. The four reactions were combined and the mixturewas diluted with water (800 mL) and dichloromethane (800 L), thenadjusted to pH=3˜4 with 0.5 N HCl solution. The organic layer wasseparated and the aqueous phase was extracted with dichloromethane (800mL). The organic layers were combined and washed with brine (1 L), thendried with anhydrous Na₂SO₄, filtered and concentrated in vacuum to givethe crude product. The crude product was purified by columnchromatography on silica gel with DCM/MeOH to give compound 29d as awhite solid.

-   Yield: 140 g, 168 mmol, 81%

Four reactions were carried out in parallel.

To a solution of compound 29d (30 g, 36 mmol, 1 eq) in THF (300 mL) wasadded (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)acetaldehyde (6.8 g, 36mmol, 1 eq) and NaBH(OAc)₃ (15.3 g, 72 mmol, 2 eq) in one portion. Afteraddition, the reaction mixture was stirred at 15-30° C. for 18 hours.TLC (DCM/MeOH=10:1, R_(f)=0.4) showed the starting material was consumedcompletely. The four reactions were combined and the mixture was dilutedwith water (2 L) and EtOAc (1.5 L). The organic layer was separated andthe aqueous phase was extracted with EtOAc (1 L). The organic layerswere combined and washed with brine (1 L), then dried with anhydrousNa₂SO₄, filtered and concentrated in vacuum to give compound 29e asyellow oil.

-   Yield: 164 g, crude

Three reactions were carried out in parallel.

To a solution of compound 29e (50 g, 49.7 mmol, 1 eq) in DCM (150 mL)was added Et₃N (25.1 g, 248 mmol, 34.4 mL, 5 eq) and Boc₂O (21.7 g, 99.4mmol, 22.8 mL, 2 eq). After addition, the reaction mixture was stirredat 15-30° C. for 12 hours. TLC (Petroleum ether/Ethyl acetate=1:1,R_(f)=0.4) showed the starting material was consumed completely. Thethree reactions were combined and the mixture was diluted with water(800 mL), then adjusted to pH=3˜4 with 0.5 N HCl solution. The organiclayer was separated and the aqueous phase was extracted withdichloromethane (800 mL). The organic layers were combined and washedwith brine (800 mL), then dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum to give the crude product. The crude product waspurified by column chromatography on silica gel with petroleumether/ethyl acetate to give compound 29f as yellow solid.

-   Yield: 80 g, 72.3 mmol, 48.5%

Three reactions were carried out in parallel.

To a solution of compound 29f (25 g, 22.6 mmol, 1 eq) in DCM (125 mL)and EtOH (300 mL) was added NH₂NH₂.H₂O (28.9 g, 565 mmol, 28 mL, 98%purity, 25 eq) in one portion. After addition, the reaction mixture wasstirred at 15-30° C. for 18 hours. TLC (Petroleum ether/Ethylacetate=1:1, R_(f)=0.03) showed the starting material was consumedcompletely. The three reactions were combined and the mixture wasdiluted with water (1 L) and dichloromethane (800 mL), then adjusted topH=3˜4 with 0.5 N HCl solution. The organic layer was separated and theaqueous phase was extracted with dichloromethane (500 mL). The organiclayers were combined and washed with brine (800 mL), then dried withanhydrous Na₂SO₄, filtered and concentrated in vacuum to give the crudeproduct. The crude product was purified by column chromatography onsilica gel with DCM/MeOH to give compound 29g as yellow oil.

-   Yield: 45 g, 46.1 mmol, 68%

Four reactions were carried out in parallel.

To a solution of compound 29g (11 g, 11.3 mmol, 1.0 eq) in THF (100 mL)was added Et₃N (3.4 g, 33.8 mmol, 4.7 mL, 3.0 eq) and 4-nitrophenylcarbonochloridate (2.5 g, 12.4 mmol, 1.1 eq). After addition, thereaction mixture was stirred at 15-30° C. for 18 hours. TLC (Petroleumether/Ethyl acetate=1:1, R_(f)=0.4) showed the starting material wasconsumed completely. The four reactions were combined and the mixturewas diluted with water (800 mL) and EtOAc (800 mL), then adjusted topH=3˜4 with 0.5 N HCl solution. The organic layer was separated and theaqueous phase was extracted with EtOAc (500 mL). The organic layers werecombined and washed with brine (800 mL), then dried with anhydrousNa₂SO₄, filtered and concentrated in vacuum to give the crude product.The crude product was purified by column chromatography on silica gelwith petroleum ether: ethyl acetate to give 29h as pale yellow stickyoil.

-   Yield: 29 g, 25.4 mmol, 56%

Example 30 Synthesis of Transient S1 PTH(1-34) Conjugate 30

Side chain protected PTH(1-34) on TCP resin having Fmoc protectedN-terminus was Fmoc deprotected according to the procedure given inMaterials and Methods. A solution of Fmoc-Ser (Trt)-OH (997 mg, 1.75mmol), PyBOP (911 mg, 1.75 mmol) and DIPEA (305 μL, 1.75 mmol) in DMF (5mL) was added to 5.0 g (0.58 mmol) of the resin. The suspension wasagitated overnight at rt. The resin was washed 10× with DMF andFmoc-deprotection was performed as described above. A solution of 29h(2.66 g, 2.33 mmol) and DIPEA (611 μL, 3.50 mmol) in DMF (5 mL) wasadded to the resin. The suspension was agitated overnight at rt. Theresin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavageof the peptide from the resin and removal of protecting groups wasachieved by adding 30 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v)TFA/EDT/TES/water/thioanisole and agitating the suspension for 1 h atrt. Crude 30 was precipitated in pre-cooled diethyl ether (−18° C.). Theprecipitate was dissolved in ACN/water and purified by RP-HPLC. Theproduct fractions were freeze-dried.

-   Yield: 168 mg (5%), 30*9 TFA-   MS: m/z 1155.92=[M+4H]⁴, (calculated monoisotopic mass for    [M+4H]⁴+=1155.85).

Example 31 Synthesis of Transient 4×10 kDa S1 PEG Conjugate 31

2.3 mL of a solution containing 30 (13 mg/mL, 30 mg, 5.31 μmol) in 8/2(v/v) water/ethanol containing 0.1% TFA (v/v) and 10 mM methionine wereadded to 3.4 mL of a solution containing PEG 2×10 kDa maleimide(Sunbright GL2-200MA, 342 mg, 15.9 μmol) in the same solvent. 0.5 MNaH₂PO₄ buffer (0.8 mL, pH 7.0) was added and the mixture was stirredfor 30 min at rt. 20 μL of TFA was added and the mixture was stored at4° C. overnight. Conjugate 31 was purified by IEX followed by RP-HPLCusing a linear gradient of solvent system A (water containing 0.2% AcOHv/v) and solvent system B (acetonitrile containing 0.2% AcOH v/v). Theproduct containing fractions were freeze-dried.

-   Yield: 161 mg (3.55 μmol, 67%) conjugate 31*9 AcOH

Example 32 In Vitro Release Kinetics of Transient 4×10 kDa PEG Conjugate31

Conjugate 31 (11 mg) was dissolved in 1 vol % acetic acid in water (1.8mL) at 0.5 mg PTHeq/mL. Buffer exchange to pH 7.4 phosphate buffer (100mM NaH₂PO₄, 10 mM L-methionine, 3 mM EDTA, 0.05% Tween-20, adjusted topH 7.4 by NaOH) was performed by SEC chromatography. The eluate wasfurther diluted with phosphate buffer to reach a concentration of 0.1 mgPTHeq/mL. The resulting solution was sterile filtered and incubated at37° C. At time points, aliquots were withdrawn and analysed by RP-HPLC.The fraction of released PTH at a particular time point was calculatedfrom the ratio of UV peak areas of liberated PTH and PEG conjugate. The% released PTH was plotted against incubation time. Curve-fittingsoftware was applied to calculate the corresponding half times ofrelease.

Results:

For conjugate 31 a release half-life time of 14.5 d was obtained.

Example 33

Pharmacodynamic Actions in Cynomolgus Monkeys During a Single Dose PK/PDStudy with Compound 18

Compound 18 was administered at 1 μg/kg to male cynomolgus monkeys (N=3)in a single subcutaneous PK/PD study assessing serum calcium (sCa)levels and urinary calcium excretion for 96 hours post-dose.

Results: Following compound 18 administration at 1 μg/kg to cynomolgusmonkeys, sCa levels remained within the normal range for 96 hourspost-dose and a clear trend for decreased urinary calcium levels overthe first 24 hours was observed.

Conclusion: At a dose maintaining sCa in the normocalcemic range, aconcurrent decrease in urinary Ca excretion was observed and compound 18hereby addresses a key unmet medical need in patients with HP.

Example 34 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate18 in Cynomolgus Monkeys

Naïve cynomolgus monkeys (2-3.5 years, 2-5 kg) (3-5 males/3-5 females)received daily subcutaneous administrations of 18 at dose levels of 0.2,0.5 and 1.5 μg PTH/kg. Blood samples were collected at; Day 1: pre-dose,4 h, 8 h, 12 h, 18 h, and 24 h post-dose, at Day 8: pre-dose, at Day 14:predose, 8h, and 12 h and at Day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, 72h, 168 h, and 336 h) and plasma was generated. Total PTH plasmaconcentrations were determined by quantification of the N-terminalsignature peptide (sequence: IQLMHNLGK) after LysC and GluC digestion aspresented earlier in Materials and Methods.

Results: Systemic exposure expressed as C_(max) and AUC increased in anapproximately dose proportional manner. Systemic exposure of Total PTHexpressed as AUC accumulated approximately 3-fold from Day 1 to Day 28.

A low mean peak-to-trough ratio of Total PTH for all dose groups wasobserved after daily subcutaneous administration in cynomolgus monkeysat Day 28 (steady state observed from Day 8).

Example 35 Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate18 in Sprague-Dawley Rats

Sprague-Dawley Crl:CD(SD) rats (initiation of dosing at 8 weeks of age)received daily subcutaneous administrations of 18 at dose levels of 10,30 and 60 μg PTH/kg for 28 days. A TK group containing of 9 males and 9females per dose group was divided into 3 subgroups with 3 rats persubgroup. Blood samples were collected up to 28 days with 3 rats persex, per sampling time point. Samples were collected at Day 1: pre-dose,4 h, 8 h, 12 h, 18 h, and 24 h post-dose, and at Day 28: 3 h, 6 h, 8 h,12 h, 18 h, 24 h, and 336 h and plasma was generated. The total PTHplasma concentrations were determined by quantification of theN-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluCdigestion as presented earlier in Materials and Methods.

Results: Systemic exposure expressed as mean C_(max) and AUC increasedin an approximately dose proportional manner. Systemic exposure of TotalPTH expressed as mean AUC accumulated 3-6 fold from Day 1 to Day 28.Systemic exposure in the female rat was approximately 2-fold higher thanin males.

A low mean peak-to-trough ratio of Total PTH for all dose groups wasobserved after daily subcutaneous administration in Sprague-Dawley ratsat Day 28 (steady state observed from Day 8).

ABBREVIATIONS

-   ACN acetonitrile-   AcOH acetic acid-   Aib 2-aminoisobutyric acid-   BMD bone mineral density-   Bn benzyl-   Boc tert-butyloxycarbonyl-   COMU    (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium    hexafluorophosphate-   cAMP cyclic adenosine monophosphate-   d day-   DBU 1,3-diazabicyclo[5.4.0]undecene-   DCC N,N′-dicyclohexylcarbodiimide-   DCM dichloromethane-   DIPEA N,N-diisopropylethylamine-   DMAP dimethylamino-pyridine-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   DTT dithiothreitol-   EDTA ethylenediaminetetraacetic acid-   eq stoichiometric equivalent-   ESI-MS electrospray ionization mass spectrometry-   Et ethyl-   Fmoc 9-fluorenylmethyloxycarbonyl-   Glu-C endoproteinase Glu-C-   h hour-   HATU O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HP hypoparathyroidism-   HPLC high performance liquid chromatography-   ivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-   LC liquid chromatography-   LTQ linear trap quadrupole-   Lys-C endoproteinase Lys-C-   LLOQ lower limit of quantification-   Mal 3-maleimido propyl-   Me methyl-   MeOH methanol-   min minutes-   Mmt monomethoxytrityl-   MS mass spectrum/mass spectrometry-   m/z mass-to-charge ratio-   OtBu tert-butyloxy-   PEG poly(ethylene glycol)-   pH potentia Hydrogenii-   PK pharmacokinetics-   Pr propyl-   PTH parathyroid hormone-   PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium    hexafluorophosphate-   Q-TOF quadrupole time-of-flight-   RP-HPLC reversed-phase high performance liquid chromatography-   rt room temperature-   sCa serum calcium-   SIM single ion monitoring-   SEC size exclusion chromatography-   sc subcutaneous-   sP serum phosphate-   t_(1/2) half life-   TCP tritylchloride polystyrol-   TES triethylsilane-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   TK toxicokinetic-   Tmob 2,4,6-trimethoxybenzyl-   TPTx thyroparathyroidectomy-   Trt triphenylmethyl, trityl-   ULOQ upper limit of quantification-   UPLC ultra performance liquid chromatography-   UV ultraviolet-   ZQ single quadrupole

1. A PTH prodrug or a pharmaceutically acceptable salt thereofcomprising a conjugate D-L, wherein -D is a PTH moiety; and -L comprisesa reversible prodrug linker moiety -L¹-, which moiety -L¹- is connectedto the PTH moiety -D through a functional group of PTH; wherein -L¹- issubstituted with -L²-Z′ and is optionally further substituted; wherein-L²- is a single chemical bond or a spacer moiety; and Z′ is awater-insoluble carrier moiety to which a multitude of moieties -L²-L¹-Dis connected.
 2. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 1, wherein -D has the sequence of SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:107, SEQ ID NO:108, SEQ IDNO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQID NO:114 or SEQ ID NO:115.
 3. The PTH prodrug or the pharmaceuticallyacceptable salt thereof of claim 1, wherein -D has the sequence of SEQID NO:51.
 4. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 1, wherein the moiety -L¹- is either conjugated to afunctional group of the side chain of an amino acid residue of -D, tothe N-terminal amine functional group or to the C-terminal carboxylfunctional group of -D or to a nitrogen atom in the backbone polypeptidechain of -D.
 5. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 1, wherein -L¹- is conjugated to a functional group ofthe side chain of a proteinogenic amino acid residue of -D selected fromthe group consisting of histidine, lysine, tryptophan, serine,threonine, tyrosine, aspartic acid, glutamic acid and arginine.
 6. ThePTH prodrug or the pharmaceutically acceptable salt thereof of claim 1,wherein -L¹- is conjugated to a functional group of the side chain of alysine of -D.
 7. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 1, wherein -L¹- is conjugated to a functional group ofthe side chain of a serine of -D.
 8. The PTH prodrug or thepharmaceutically acceptable salt thereof of claim 1, wherein -L¹- isconjugated to the N-terminal amine functional group of -D.
 9. The PTHprodrug or the pharmaceutical acceptable salt thereof of claim 1,wherein -L¹- is connected to -D through a linkage selected from thegroup consisting of amide, ester, carbamate, acetal, aminal, imine,oxime, hydrazone, disulfide and acylguanidine.
 10. The PTH prodrug orthe pharmaceutical acceptable salt thereof of claim 1, wherein -L¹- is areversible prodrug linker from which the PTH is released in its freeform.
 11. The PTH prodrug or the pharmaceutical acceptable salt thereofof claim 1, wherein wherein -L¹- is of formula (IV):

wherein the dashed line indicates attachment to -D and whereinattachment is through a functional group of -D selected from the groupconsisting of —OH, —SH and —NH₂; m is 0 or 1; at least one or both of—R¹ and —R² is/are independently of each other selected from the groupconsisting of —CN, —NO₂, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkenyl, optionallysubstituted alkynyl, —C(O)R³, —S(O)R³, —S(O)₂R³, and —SR⁴, one and onlyone of —R¹ and —R² is selected from the group consisting of —H,optionally substituted alkyl, optionally substituted arylalkyl, andoptionally substituted heteroarylalkyl; —R³ is selected from the groupconsisting of —H, optionally substituted alkyl, optionally substitutedaryl, optionally substituted arylalkyl, optionally substitutedheteroaryl, optionally substituted heteroarylalkyl, —OR⁹ and —N(R⁹)₂;—R⁴ is selected from the group consisting of optionally substitutedalkyl, optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, and optionally substitutedheteroarylalkyl; each —R⁵ is independently selected from the groupconsisting of —H, optionally substituted alkyl, optionally substitutedalkenylalkyl, optionally substituted alkynylalkyl, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroaryl and optionally substituted heteroarylalkyl; —R⁹is selected from the group consisting of —H and optionally substitutedalkyl; —Y— is absent and —X— is —O— or —S—; or —Y— is —N(Q)CH₂— and —X—is —O—; Q is selected from the group consisting of optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedarylalkyl, optionally substituted heteroaryl and optionally substitutedheteroarylalkyl; optionally, —R¹ and —R² may be joined to form a 3 to8-membered ring; and optionally, both —R⁹ together with the nitrogen towhich they are attached form a heterocyclic ring; wherein -L¹- issubstituted with -L²-Z′ and wherein -L¹- is optionally furthersubstituted.
 12. The PTH prodrug or the pharmaceutical acceptable saltthereof of claim 1, wherein -L²- is a chemical bond.
 13. The PTH prodrugor the pharmaceutical acceptable salt thereof of claim 1, wherein -L²-is a spacer moiety.
 14. The PTH prodrug or the pharmaceuticallyacceptable salt thereof of claim 1, wherein -L²- selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y1))—,—S(O)₂N(R^(y1))—, —S(O)N(R^(y1))—, —S(O)₂—, —S(O)—,—N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—,—N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl,and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally substituted with one or more —R^(y2), which arethe same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—,—S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R^(y3))—; —R^(y1) and —R^(y1a)are independently of each other selected from the group consisting of—H, -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein -T, C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally substituted withone or more —R^(y2), which are the same or different, and wherein C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of -T-, —C(O)O—,—O—, —C(O)—, —C(O)N(R^(y4))—, —S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—,—S(O)₂—, —S(O)—, —N(R^(y4))S(O)₂N(R^(y4a))—, —S—, —N(R^(y4))—,—OC(OR^(y4))(R^(y4a))—, —N(R^(y4))C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R^(y2), which are the same or different; each —R^(y2) is independentlyselected from the group consisting of halogen, —CN, oxo (═O),—COOR^(y5), —OR^(y5), —C(O)R^(y5), —C(O)N(R^(y5)R^(y5a)),—S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)), —S(O)₂R^(y5),—S(O)R^(y5), —N(R^(y5))S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(y5)R^(y5a)), —NO₂, —OC(O)R^(y5), —N(R^(y5))C(O)R^(y5a),—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5a),—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and each —R^(y3), —R^(y3a), —R^(y4),—R^(y4a), —R^(y5), R^(y5a) and —R^(y5b) is independently selected fromthe group consisting of —H, and C₁₋₆ alkyl, wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent.
 15. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 1, wherein -L²- is a C₁₋₂₀ alkyl chain, which isoptionally interrupted by one or more groups independently selected from—O—, -T- and —C(O)N(R^(y1))—; and which C₁₋₂₀ alkyl chain is optionallysubstituted with one or more groups independently selected from —OH, -Tand —C(O)N(R^(y6)R^(y6a)); wherein —R^(y1), —R^(y6), —R^(y6a) areindependently selected from the group consisting of H and C₁₋₄ alkyl andwherein T is selected from the group consisting of phenyl, naphthyl,indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to 10-memberedheterocyclyl, 8- to 11-membered heterobicyclyl, 8-to 30-memberedcarbopolycyclyl, and 8- to 30-membered heteropolycyclyl.
 16. The PTHprodrug or the pharmaceutically acceptable salt thereof of claim 1,wherein -L²- has a molecular weight in the range of from 14 g/mol to 750g/mol.
 17. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 1, wherein -L²- has a chain length of 1 to 20 atoms.18. The PTH prodrug or the pharmaceutically acceptable salt thereof ofclaim 1, wherein -Z′ is a hydrogel.
 19. The PTH prodrug or thepharmaceutically acceptable salt thereof of claim 18, wherein thehydrogel comprises a polymer selected from the group consisting of2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.
 20. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 18, wherein the hydrogel comprises PEG or hyaluronicacid.
 21. The PTH prodrug or the pharmaceutically acceptable saltthereof of claim 18, wherein the hydrogel comprises PEG.
 22. Apharmaceutical composition comprising at least one PTH prodrug or thepharmaceutically acceptable salt thereof of claim 1 and at least oneexcipient.
 23. The pharmaceutical composition of claim 22, wherein thepharmaceutical composition has a pH ranging from and including pH 4 topH
 6. 24. A method of treating, controlling, delaying or preventing in amammalian patient in need of the treatment of one or more diseases whichcan be treated with PTH, comprising the step of administering to saidpatient in need thereof a therapeutically effective amount of the PTHprodrug or the pharmaceutically acceptable salt thereof of claim
 1. 25.The method of claim 24, wherein the disease is selected from the listconsisting of hypoparathyroidism, hyperphosphatemia, osteoporosis,fracture repair, osteomalacia, osteomalacia and osteoporosis in patientswith hypophosphatasia, steroid-induced osteoporosis, male osteoporosis,arthritis, osteoarthritis, osteogenesis imperfect, fibrous dysplasia,rheumatoid arthritis, Paget's disease, humoral hypercalcemia associatedwith malignancy, osteopenia, periodontal disease, bone fracture,alopecia, chemotherapy-induced alopecia, and thrombocytopenia.
 26. Themethod of claim 24, wherein the disease is hypoparathyroidism.
 27. Themethod of claim 24, wherein the mammalian patient is a human patient.28. The method of claim 24, wherein the time period between twoconsecutive administrations is one week.
 29. The method of claim 24,wherein the prodrug or the pharmaceutically acceptable salt thereof isadministered via injection.
 30. The method of claim 24, wherein theprodrug or the pharmaceutically acceptable salt thereof is administeredvia subcutaneous injection.
 31. The method of claim 24, wherein thesubcutaneous injection is with a pen injector.